WO2013180406A1 - Primer set for detecting foot and mouth disease according to serum type, pcr device using same, and method for detecting foot and mouth disease by using same - Google Patents

Primer set for detecting foot and mouth disease according to serum type, pcr device using same, and method for detecting foot and mouth disease by using same Download PDF

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Publication number
WO2013180406A1
WO2013180406A1 PCT/KR2013/004189 KR2013004189W WO2013180406A1 WO 2013180406 A1 WO2013180406 A1 WO 2013180406A1 KR 2013004189 W KR2013004189 W KR 2013004189W WO 2013180406 A1 WO2013180406 A1 WO 2013180406A1
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Prior art keywords
pcr
light
syto
heater
foot
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PCT/KR2013/004189
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French (fr)
Korean (ko)
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김성우
김선진
이세현
류호선
이동훈
김덕중
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나노바이오시스(주)
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Publication of WO2013180406A1 publication Critical patent/WO2013180406A1/en

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to a detection device and a detection method for detecting whether foot and mouth disease (FMD).
  • FMD foot and mouth disease
  • Foot-and-mouth disease is a viral bullous disease that infects animals with two hoofs, such as cattle and pigs, and is characterized by rapid replication and transmission. Because foot-and-mouth disease is a typical livestock disease, its economic importance is managed by the International Water Services Bureau (OIE), and large outbreaks in cross-border imports and exports of livestock animals occur when foot-and-mouth disease develops. Animals infected with foot-and-mouth disease develop a high fever, but after two days, the heat subsides, the blisters in the mouth cause a lot of foamy and sticky saliva, and the hooves develop blisters and limping.
  • OIE International Water Services Bureau
  • real-time PCR (Real-time Polymerase Chain Reaction) is a method for observing the increase of the PCR amplification product in real time every cycle of the PCR, it is a method for interpreting by the detection and quantification of the fluorescent material reacted with the PCR amplification product.
  • Real-time PCR does not require any additional electrophoresis, it is highly accurate, sensitive, has high reproducibility, and is automated, compared to conventional PCR, which is stained on gel after final step to identify PCR amplification products after electrophoresis.
  • One embodiment of the present invention is to provide a primer set, a series of foot-and-mouth disease detection device and a detection method capable of simultaneously specifically amplifying genes related to the cause virus of each type of foot-and-mouth disease.
  • the first embodiment of the present invention comprises foot-and-mouth virus A, which comprises a primer comprising 15 or more consecutive nucleotides of SEQ ID NO: 1 and a primer comprising 15 or more consecutive nucleotides of SEQ ID NO: Genbank ID number: NC011450), foot and mouth virus O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451 ), And foot and mouth virus SAT 2 (Genbank ID number: NC011452) provides a primer set for detecting foot and mouth disease (Foot and mouth disease (FMD)) for detecting one or more genes selected from the group consisting of.
  • FMD foot and mouth disease
  • a second embodiment of the present invention comprises foot-and-mouth virus A, which comprises a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 3 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 4 Genbank ID number: NC011450), foot and mouth virus O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451 ), And foot and mouth virus SAT 2 (Genbank ID number: NC011452) provides a primer set for detecting foot and mouth disease (Foot and mouth disease (FMD)) for detecting one or more genes selected from the group consisting of.
  • FMD foot and mouth disease
  • a third embodiment of the present invention is the first plate; A second plate disposed on the first plate and having one or more reaction channels; And a third plate disposed on the second plate, the third plate having an inlet and an outlet connected to both ends of the at least one reaction channel and configured to be openable and closed. It provides a PCR (Polymerase Chain Reaction) chip comprising a primer set according to the first embodiment.
  • PCR Polymerase Chain Reaction
  • the first plate and the third plate is polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate, PMMA) ), Polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof
  • the second plate is made of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (CCO), polyamide (PA), polyethylene (polyethylene, PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) ), Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate ( polybutylene terephthalate
  • the PCR chip is implemented in a plastic material, it may be implemented to have a light transmission.
  • the PCR chip may further comprise a mixture comprising dATP, dCTP, dGTP, and dTTP, DNA polymerase and detectable label in the one or more reaction channels.
  • the detectable label is Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680 , Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO
  • a fourth embodiment of the present invention includes a first thermal block disposed on a substrate; A second thermal block spaced apart from the first thermal block on the substrate; And a chip holder movable left, right and / or up and down by the driving means over the first row block and the second row block, and equipped with a PCR chip according to the third embodiment of the present invention.
  • a light source is further disposed between the first column block and the second column block, and a light detector for detecting light emitted from the light source is further disposed on the chip holder, or A light detector for detecting light emitted from the light source may be further disposed between the first and second heat blocks, and a light source may be further disposed on the chip holder.
  • a fifth embodiment of the present invention includes a substrate, a heat generating layer including conductive nanoparticles disposed on the substrate, an insulating protective layer disposed on the heat generating layer, and an electrode disposed in connection with the heat generating layer, but is light-transparent
  • a light transmissive thermal block implemented to have; And it provides a PCR device comprising a PCR chip according to a third embodiment of the present invention, arranged to be in contact with the upper surface of the light transmitting thermal block.
  • the substrate is a light-transmissive glass or plastic material
  • the conductive nanoparticles included in the heat generating layer is an oxide semiconductor material or In, Sb, Al, Ga, C and It is a material to which impurities selected from the group consisting of Sn is added
  • the insulating protective layer is selected from the group consisting of dielectric oxide, perylene, nanoparticles and polymer film
  • the electrode is a metal material, conductive epoxy, conductive paste, solder And it may be selected from the group consisting of a conductive film.
  • the PCR device may further include a light providing unit operably arranged to provide light to a PCR chip disposed in the chip contact unit, and a light detection unit operatively arranged to receive light emitted from the PCR chip disposed in the chip contact unit. It may further include.
  • a heater group including one or more heaters, two or more heater groups, and the two or more heater groups include two or more heater units spaced apart from each other so that mutual heat exchange does not occur.
  • a thermal block having a contact surface of a PCR chip containing at least one target sample thereon; An electrode unit having an electrode connected to supply electric power to heaters provided in the thermal block; And it provides a PCR device comprising a PCR chip according to a third embodiment of the present invention, arranged to be in contact with the heat block to enable heat exchange with one or more heaters provided in the heat block.
  • the thermal block may include two to four heater groups.
  • the thermal block has two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature.
  • the thermal block includes three heater groups, wherein the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group has the PCR extension step temperature.
  • the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature
  • the first heater The group may maintain the PCR extension step temperature
  • the second heater group may maintain the PCR denaturation step temperature
  • the third heater group may maintain the PCR annealing step temperature.
  • the thermal block may be implemented to have light transmittance.
  • the heater provided in the heat block may include a light transmitting heating element.
  • the apparatus may further include a power supply unit for supplying power to the electrode unit and a pump arranged to provide a positive pressure or a negative pressure to control a flow rate and a flow rate of the fluid flowing in the one or more reaction channels.
  • a light source is disposed between the first heater and the second heater, a power supply for supplying power to the electrode portion, a positive pressure to control the flow rate and flow rate of the fluid flowing in the at least one reaction channel or
  • the apparatus may further include a pump disposed to provide a negative pressure, and a light detector for detecting light emitted from the light source.
  • the light providing unit may further include a light detecting unit disposed to receive the light emitted from the PCR chip.
  • a seventh embodiment of the present invention includes the steps of performing a PCR by introducing a target sample suspected of foot and mouth infection into the one or more reaction channels of the PCR chip according to the third embodiment of the present invention; And it provides a foot-and-mouth disease detection method comprising the step of confirming the presence or absence of foot-and-mouth virus in the target sample from the PCR results.
  • the step of performing PCR is a PCR device according to a third embodiment of the present invention, a PCR device according to the fourth embodiment of the present invention, and a PCR according to the fifth embodiment of the present invention. And may be performed in a PCR device selected from the group consisting of devices.
  • a primer set capable of simultaneously specifically amplifying a gene related to a cause virus of each type of foot-and-mouth disease, a PCR chip comprising the same, a PCR device including the same, and a foot-and-mouth disease detecting method using the same
  • the outbreak of foot-and-mouth disease can be accurately and quickly confirmed at a low cost, thereby greatly contributing to preventing the spread of foot-and-mouth disease and taking prompt response thereto.
  • FIG. 1 is a diagram of a PCR chip according to a third embodiment of the present invention.
  • FIG. 2 is a diagram of a PCR chip according to a third embodiment of the present invention in which a double-sided adhesive, a thermoplastic resin, or a thermosetting resin is treated.
  • 3A to 3C are diagrams of a PCR device according to a fourth embodiment of the present invention.
  • 4A to 4I are diagrams illustrating a PCR device according to a fifth embodiment of the present invention.
  • 5A to 5D are diagrams of a PCR device according to a sixth embodiment of the present invention.
  • Figures 6 to 11 show the results of PCR comparison of the foot-and-mouth disease virus plasmid samples for each serotype by using a third-party PCR device and a PCR device according to a fourth embodiment of the present invention.
  • a first embodiment of the present invention is a primer comprising at least 15 consecutive nucleotides of the base sequence of SEQ ID NO: 1 (CAC GCC GTG GGA CYA THC AGG A) and the base sequence of SEQ ID NO: 2 (GGG YTC RAA GAG RCG CCG GTA Foot-and-mouth virus A (Genbank ID number: NC011450), foot-and-mouth virus O (Genbank ID number: NC004004), foot-and-mouth virus C (Genbank ID number: NC002554), foot-and-mouth disease, consisting of primers comprising at least 15 consecutive nucleotides of Y)
  • serotype to detect one or more selected genes from the group consisting of virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451), and foot and mouth virus SAT 2 (Genbank ID number: NC011452)
  • FMD foot and mouth disease
  • the second embodiment of the present invention is a sequence of
  • the primers are single-stranded oligonucleotides that can serve as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers at suitable temperatures.
  • suitable conditions ie, four different nucleoside triphosphates and polymerases
  • Suitable lengths of the primers are typically 15 to 30 nucleotides, depending on various factors, such as temperature and the use of the primer. Short primers may generally require lower temperatures to form a hybridization complex that is sufficiently stable with the template.
  • the forward primer and the reverse primer mean primers that bind to the 3 'end and the 5' end of a predetermined portion of the template to be amplified by the polymerase chain reaction.
  • the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range that can hybridize with the template to perform the primer-specific function. Therefore, the primer sets according to the first to second embodiments of the present invention do not need to have a sequence that is completely complementary to the nucleotide sequence that is a template, and sufficient complementarity within a range capable of hybridizing to this sequence to act as a primer. It is enough to have.
  • the design of such primers can be easily carried out by those skilled in the art by referring to the nucleotide sequence of the polynucleotide to be a template, for example, using a primer design program (for example, PRIMER 3, VectorNTI program). have.
  • the primer according to an embodiment of the present invention is hybridized or annealed to one site of the template to form a double-chain structure.
  • Conditions for nucleic acid hybridization suitable for forming such double chain structures are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington, DC (1985).
  • the primer may comprise 15 or more consecutive nucleotides in the base sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer is the base of any one of SEQ ID NO: 1 and SEQ ID NO: 2 It may be an oligonucleotide having a sequence, wherein the primer may comprise at least 15 consecutive nucleotides in the base sequence of any one of SEQ ID NO: 3 and SEQ ID NO: 4, the primer is SEQ ID NO: 3 and SEQ ID NO: 4 It may be an oligonucleotide having any one of the base sequence.
  • the size of the PCR product of the primer set according to the first to second embodiments of the present invention is 90 to 120 bp (base pair), preferably 92 bp (base pair), the annealing temperature is 70 to 72 °C, preferably Preferably, primers of SEQ ID NOs: 1 and 3 are 72.7 ° C., primers of SEQ ID NOs: 2 and 4 are 71.5 ° C., GC% is 50-65%, preferably primers of SEQ ID NOs: 1 and 3 are 63.6 ° C., SEQ ID NOs: 2 and The primer of 4 was designed at 63.6 ° C.
  • the method of detecting foot-and-mouth disease using the primer set is as follows. First, PCR is performed by introducing a target sample suspected of foot and mouth infection into the one or more reaction channels of a PCR chip which will be described in detail below.
  • Subject sample refers to a sample (sample or sample solution) of an individual expected to be infected with foot and mouth disease, and includes, but is not limited to, for example, cultured cells, blood, saliva, and the like.
  • the target sample suspected of foot-and-mouth disease infection may be assumed to synthesize cDNA from the RNA of the sample.
  • Foot-and-mouth disease viruses take RNA as a genome, perform reverse transcription from the virus's genomic RNA, and synthesize cDNA from it to obtain the template DNA necessary to perform PCR for detecting it.
  • Reverse transcription reactions can be carried out through a variety of known reverse transcriptase enzymes, for example, the SuperScript series from Invtrogen, and kits comprising the same.
  • the synthesized cDNA is introduced into a PCR chip to perform real time PCR. Therefore, the presence or absence of foot-and-mouth virus in the target sample can be confirmed from the PCR result.
  • the step of performing PCR may be performed in a series of PCR devices to be described in detail below.
  • PCR amplification is performed from a curve that is detected by detecting a fluorescent labeling factor labeled on an amplification product during PCR. This can be confirmed by calculating the Ct value, which is the number of cycles when the product is amplified by a certain amount.
  • the calculation of the Ct value may be automatically performed by a program installed in a real-time PCR apparatus.
  • dNTP deoxyribonucleotide triphosphates
  • dATP deoxyribonucleotide triphosphates
  • dCTP dCTP
  • dGTP dGTP
  • a mixture comprising dTTP, a DNA polymerase, a detectable label, and a PCR buffer.
  • the DNA polymerase may be, for example, a heat stable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus furiosus (Pfu).
  • the detectable label is Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2 , Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43 , SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SY
  • the PCR buffer is a compound that is added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction.
  • buffers are known, for example Tris, Tricine, MOPS, or HEPES, but is not limited thereto.
  • the PCR chip 10 includes an inlet 15 for introducing the sample solution, an outlet 16 for discharging the sample solution having completed the nucleic acid amplification reaction, and a sample solution containing the nucleic acid to be amplified.
  • the reaction channel 14 may be included.
  • the PCR chip 10 When the PCR chip 10 contacts the thermal block of the PCR device, heat generated in the thermal block is transferred to the PCR chip 10, and a sample included in the reaction channel 14 of the PCR chip 10. The solution may be heated or cooled to maintain a constant temperature.
  • the PCR chip 10 may have a flat shape as a whole, but is not limited thereto.
  • the PCR chip 10 may be disposed in contact with the thermal block while being mounted on the chip holder of the PCR device.
  • the arrangement of the PCR chip 10 on one surface of the column block includes contact arrangement of the PCR chip 10 with the column block while being mounted on the chip holder.
  • the PCR chip 10 is implemented with a plastic material, it may be implemented to have a light transmission.
  • the PCR chip 10 may use a plastic material to increase the heat transfer efficiency only by adjusting the thickness of the plastic, and the manufacturing process may be simplified to reduce the manufacturing cost.
  • the PCR chip 10 may be provided with light transmittance as a whole, light can be directly irradiated in a state in which it is disposed on one surface of the heat block, thereby measuring and analyzing nucleic acid amplification and amplification degree in real time. .
  • the first plate 11 is disposed on the bottom surface of the second plate 12.
  • the first plate 11 is bonded to the lower surface of the second plate 920 so that the one or more reaction channels 14 form a kind of PCR reaction chamber.
  • the first plate 11 may be made of various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from.
  • the hydrophilic material 17 is treated on the upper surface of the first plate 11 to perform PCR smoothly.
  • a single layer comprising hydrophilic material 17 may be formed on the first plate 11.
  • the hydrophilic material may be a variety of materials, but preferably may be selected from the group consisting of carboxyl group (-COOH), amine group (-NH2), hydroxy group (-OH), and sulfone group (-SH), Treatment of the hydrophilic material can be carried out according to methods known in the art.
  • the second plate 12 is disposed on an upper surface of the first plate 11.
  • the second plate 920 includes one or more reaction channels 14.
  • the reaction channel 14 is connected to portions corresponding to the inlet 15 and the outlet 16 formed in the third plate 13 to form a kind of PCR reaction chamber. Therefore, PCR is performed after the sample solution to be amplified is introduced into the reaction channel 921.
  • the reaction channel 14 may be present in two or more according to the purpose and scope of use of the PCR apparatus, and according to FIG. 1, six reaction channels 14 are illustrated.
  • the second plate 12 may be made of various materials, but preferably, polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (cycloolefin copolymer, COC) , Polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (polybutylene terephthalate) , PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), and combinations thereof It is chosen or a thermoplastic resin may be a thermosetting resin material.
  • the thickness of the second plate 12 may vary, but may be selected from 100 ⁇ m to 200 ⁇ m.
  • the width and length of the reaction channel 14 may vary, but preferably the width of the reaction channel 14 is selected from 0.5 mm to 3 mm, the length of the through-opening channel 14 is 20 can be selected from mm to 40 mm.
  • the inner wall of the second plate 12 may be coated with a material such as silane-based and Bovine Serum Albumin (BSA) to prevent DNA and protein adsorption.
  • BSA Bovine Serum Albumin
  • the third plate 13 is disposed on the second plate 12.
  • the third plate 13 has an inlet 15 formed in one region on one or more reaction channels 921 formed in the second plate 12 and an outlet 16 formed in the other region.
  • the inlet 15 is a portion into which a sample solution containing a nucleic acid to be amplified is introduced.
  • the outlet 16 is a portion where the sample solution and the like flows out after the PCR is completed.
  • the third plate 13 covers one or more reaction channels 14 formed in the second plate 12, which will be discussed below, wherein the inlet 15 and outlet 16 are the reaction channels 14 ) Will serve as the inlet and outlet.
  • the third plate 13 may be made of various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from.
  • the inlet 15 may have various sizes, but preferably may be selected from 1.0 mm to 3.0 mm in diameter.
  • the outlet 16 may have a variety of sizes, but preferably may be selected from 1.0 mm to 1.5 mm in diameter.
  • the inlet part 15 and the outlet part 16 are provided with separate cover means (not shown), so that the sample solution leaks when PCR is performed on the sample solution in the reaction channel 14. It can prevent.
  • the cover means may be implemented in various shapes, sizes or materials.
  • the thickness of the third plate may vary, but may preferably be selected from 0.1 mm to 2.0 mm.
  • the inlet 15 and the outlet 16 may be present at least two.
  • the PCR chip 10 forms a inlet 15 and an outlet 16 through mechanical processing to provide a third plate 13; From the portion corresponding to the inlet portion 15 of the third plate 13 to the plate portion having a size corresponding to the lower surface of the third plate 13 to the outlet portion 16 of the third plate 13 Forming one or more reaction channels 14 by mechanical machining to the corresponding portion to provide a second plate 12; Providing a first plate (11) by forming a surface made of a hydrophilic material (17) through surface treatment on an upper surface of a plate having a size corresponding to a lower surface of the second plate (12); And bonding the lower surface of the third plate 13 to the upper surface of the second plate 12 through a bonding process, and the lower surface of the second plate 12 to the upper portion of the first plate 11.
  • the inlet 15 and outlet 16 of the third plate 13 and the reaction channel 14 of the second plate 12 are injection molded, hot-embossing and casting. ), And a processing method selected from the group consisting of laser ablation.
  • the hydrophilic material 17 on the surface of the first plate 11 may be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application and are known in the art. Can be performed according to.
  • the lower surface of the third plate 13 and the upper surface of the second plate 12, and the lower surface of the second plate 12 and the upper surface of the first plate 11 are thermally bonded, It can be adhered by ultrasonic fusion, solvent bonding processes and can be carried out according to methods known in the art.
  • a double-sided adhesive or a thermoplastic or thermosetting resin 18 may be treated between the third plate 13 and the second plate 12 and between the second plate 12 and the third plate 13.
  • the PCR device is a device for use in PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific base sequence.
  • PCR Polymerase Chain Reaction
  • a PCR device for amplifying deoxyribonucleic acid (DNA) having a specific nucleotide sequence may be used to heat a sample solution containing double stranded DNA to a specific temperature, for example about 95 ° C.
  • a denaturing step of separating DNA into strands providing an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified in the sample solution, and a specific temperature with the separated single strand of DNA.
  • an annealing step in which the primer is coupled to a specific base sequence of the single strand of DNA by cooling to 55 ° C. to form a partial DNA-primer complex, and the sample solution is titrated after the annealing step.
  • Temperature based on primers of the partial DNA-primer complex by DNA polymerase for example at 72 ° C.
  • a PCR device By performing an extension (or amplification) step of forming a double strand of DNA and repeating the three steps, for example, 20 to 40 times, the DNA having the specific base sequence can be exponentially amplified. have. Also, in some cases, the PCR device may simultaneously perform the annealing step and the extension (or amplification) step, and in this case, the PCR device may perform two steps including the extension step and the annealing and extension (or amplification) step. By performing, the first circulation may be completed. Therefore, in the present specification, a PCR device refers to a device including modules for performing the above steps, and detailed modules not described herein are all provided in the prior art and obvious range for performing PCR. It is assumed that you are doing.
  • a PCR device includes a first row block 100a disposed on a substrate 400a; A second thermal block 200a spaced apart from the first thermal block 100a on the substrate 400a; And move left, right, and / or up and down by the driving means 500a over the first row block 100a and the second row block 200a, and the PCR chip 10 according to the third embodiment of the present invention is And a mounted chip holder 300a.
  • the substrate 400a does not change its physical and / or chemical properties due to heating and temperature maintenance of the first thermal block 100a and the second thermal block 200a, and the first thermal block 100a and the first thermal block 100a and the second thermal block 200a do not change. It includes all materials having a material such that mutual heat exchange does not occur between the two heat blocks 200a.
  • the substrate 400a may include or be made of a material such as plastic.
  • the first row block 100a and the second row block 200a are for maintaining a temperature for performing a denaturation step, annealing step and extension (or amplification) step for amplifying the nucleic acid.
  • the first thermal block 100a and the second thermal block 200a may include or be operably connected with various modules for providing and maintaining the required temperature required for the respective steps. . Therefore, when the chip holder 300a on which the PCR chip 10 is mounted is in contact with one surface of each of the row blocks 100a and 200a, the first row block 100a and the second row block 200a are Since the contact surface with the PCR chip 10 can be heated and maintained at a temperature as a whole, the sample solution in the PCR chip 10 can be heated and maintained at a uniform temperature.
  • the temperature change rate in the single heat block is within a range of 3 to 7 ° C per second, whereas a PCR device including two heat blocks according to a third embodiment of the present invention
  • the rate of temperature change in each of the thermal blocks 100a and 200a may be within a range of 20 to 40 ° C. per second to greatly shorten the PCR progress time.
  • Hot wires may be disposed in the first row block 100a and the second row block 200a.
  • the heating wire may be operably connected with various heat sources to maintain a temperature for performing the denaturing, annealing and extending (or amplifying) steps, and may be operably connected with various temperature sensors for monitoring the temperature of the heating wire.
  • the heating wires are vertically and / or horizontally based on the center point of the surface of each of the heat blocks 100a and 200a in order to maintain a constant internal temperature of the first and second heat blocks 100a and 200a. It may be arranged to be symmetrical. The arrangement of the hot wires symmetrical in the vertical direction and / or the horizontal direction may vary.
  • a thin film heater (not shown) may be disposed in the first thermal block 100a and the second thermal block 200a.
  • the thin-film heater is vertically and / or horizontally based on a center point of each of the heat block 100a and 200a in order to maintain a constant internal temperature of the first and second heat blocks 100a and 200a. May be spaced apart at regular intervals.
  • the arrangement of the thin film heater that is constant in the vertical and / or horizontal directions may vary.
  • the first heat block 100a and the second heat block 200a may include a metal material, for example, aluminum or may be made of aluminum for even heat distribution and rapid heat transfer over the same area.
  • the first thermal block 100a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps.
  • the first row block 100a of the PCR apparatus according to the third embodiment of the present invention may maintain 50 ° C. to 100 ° C., and preferably, the denaturation step is performed in the first row block 100 a.
  • the temperature may be maintained at 90 ° C. to 100 ° C., preferably at 95 ° C., and may be 55 ° C. to 75 ° when the annealing and extension (or amplification) steps are performed in the first thermal block 100 a.
  • °C can be maintained, preferably 72 °C.
  • the temperature of the denaturation step or the annealing and extension (or amplification) step is not limited thereto.
  • the second row block 200a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps.
  • the second row block 200a of the PCR apparatus according to the third embodiment of the present invention may maintain 90 ° C. to 100 ° C. when the denaturation step is performed in the second row block 200 a.
  • the temperature may be maintained at 95 ° C., and may be maintained at 55 ° C. to 75 ° C., preferably at 72 ° C., when the annealing and extension (or amplification) steps are performed in the second heat block.
  • the temperature of the denaturation step or the annealing and extension (or amplification) step is not limited thereto. Therefore, according to the third embodiment of the present invention, the first row block 100a may maintain the denaturing temperature of the PCR, and if the denaturation temperature is lower than 90 ° C, the nucleic acid is a template of the PCR. When the denaturation occurs, the efficiency is low, the PCR efficiency may not be reduced, or the reaction may not occur.
  • the denaturation step temperature is higher than 100 ° C., the enzyme used for PCR loses activity, and thus, the denaturation step temperature is 90 ° C. to 100 ° C. And preferably 95 ° C.
  • the second row block 200a may maintain annealing / extension temperature of annealing and extension (or amplification) of PCR. If the extension (or amplification) step temperature is lower than 55 ° C., the specificity of the PCR product may be degraded. If the annealing and extension (or amplification) step temperature is higher than 74 ° C., the PCR may not occur. Since the efficiency is lowered, the annealing and extension (or amplification) step temperature may be 55 ° C to 75 ° C, preferably 72 ° C.
  • the first thermal block 100a and the second thermal block 200a may be spaced apart from each other at a predetermined distance such that mutual heat exchange does not occur. Accordingly, since the heat exchange does not occur between the first heat block 100a and the second heat block 200a, in the nucleic acid amplification reaction that may be significantly affected by minute temperature changes, the denaturation step and the Accurate temperature control of the annealing and extension (or amplification) steps is possible.
  • the PCR device may move left and right and / or up and down by the driving means 500a on the first row block 100a and the second row block 200a, and the PCR chip 10 may be used.
  • the mounted chip holder 300a is included.
  • the chip holder 300a is a module in which the PCR chip 10 is mounted to the PCR device.
  • the inner wall of the chip holder 300a is fixedly mounted to the outer wall of the PCR chip 10 so that the PCR chip 10 is not separated from the chip holder 300a when the nucleic acid amplification reaction is performed by the PCR apparatus. It may have a shape and structure for.
  • the chip holder 300a is operably connected to the driving means 500a.
  • the PCR chip 10 may be detachable to the chip holder (300a).
  • the driving means 500a is any means for allowing the chip holder 300a on which the PCR chip 10 is mounted to move left and right and / or up and down over the first row block 100a and the second row block 200a. It includes. By the left and right movement of the driving means 500a, the chip holder 300a on which the PCR chip 10 is mounted is capable of reciprocating between the first row block 100a and the second row block 200a. By the vertical movement of the driving means 500a, the chip holder 300a on which the PCR chip 10 is mounted may contact and be separated from the first row block 100a and the second row block 200a. have.
  • the left and right and / or vertical movement of the driving means 500a may be controlled by a control means (not shown), which is operably disposed inside or outside the PCR device, and the control means may be modified with a modification step of PCR. It is possible to control the contact and separation between the chip holder 300a on which the PCR chip 10 is mounted and the first row block 100a and the second row block 200a for annealing and extending (or amplifying) the step. have.
  • Figure 3b shows each step of the nucleic acid amplification reaction by the movement of the chip holder of the PCR device according to the fourth embodiment of the present invention.
  • the nucleic acid amplification reaction by the PCR device is based on the following steps. First, a nucleic acid, such as double-stranded DNA, oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, DNA polymerase, deoxyribonucleotide triphosphates (dNTP) in the PCR chip 10. A sample solution including a PCR buffer is introduced, and the PCR chip 10 is mounted on the chip holder 300a.
  • a nucleic acid such as double-stranded DNA
  • oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified
  • DNA polymerase DNA polymerase
  • dNTP deoxyribonucleotide triphosphates
  • the first heat block 100a is heated and maintained at a temperature for the modification step, for example, 90 ° C. to 100 ° C., preferably at 95 ° C.
  • the PCR chip 10 is moved downward by controlling the connecting member 520a of the driving means 500a to move the chip holder 300a on which the PCR chip 10 is mounted to the first row block. 100a) to perform the first denaturation step of PCR (step x).
  • the PCR chip 10 is moved upward by controlling the connecting member 520a of the driving means 500a, so that the chip holder 300a on which the PCR chip 10 is mounted is moved to the first row block ( Separating from 100a) to end the first denaturation step of PCR, and controlling the connecting member 520a of the driving means 500a to move the PCR chip 10 above the second row block 200a. Do it (step y). Subsequently, the PCR chip 10 is moved downward by controlling the connecting member 520a of the driving means 500a to move the chip holder 300a on which the PCR chip 10 is mounted to the second row block ( 100a) to perform the first annealing and extension (or amplification) step of the PCR (step z).
  • the PCR chip 10 is moved upward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a on which the PCR chip 10 is mounted is moved to the second row block ( 100a) to terminate the first annealing and extension (or amplification) step of the PCR, and control the connecting member 520a of the driving means 500a to control the PCR chip 10 by the first row block 100a.
  • the nucleic acid amplification reaction is performed by repeating the steps x, y, and z after moving up to (circulation step).
  • Figure 3c shows the step of observing the nucleic acid amplification reaction in real time using a PCR device according to a fourth embodiment of the present invention.
  • a light source 700a is further disposed between the first row block 100a and the second row block 200a, and the light source 700a is disposed on the chip holder 300a.
  • An optical detection unit 800a for detecting light emitted from the light emitting device is further disposed, or between the first thermal block 100a and the second thermal block 200a for detecting the light emitted from the light source 700a.
  • the light detector 800a may be further disposed, and the light source 700a may be further disposed on the chip holder 300a.
  • the light detector 800a may be disposed on the driving means 500a, and the through means 530a may be disposed in the driving means 900a to allow the light emitted from the light source 700a to pass therethrough.
  • the PCR chip 10 may be a light transmissive material, specifically, a light transmissive plastic material.
  • the nucleic acid amplification reaction can be detected in real time in the PCR chip 10 during the nucleic acid amplification reaction by the PCR apparatus 1.
  • a separate fluorescent substance may be further added to the sample solution introduced into the PCR chip 10.
  • the light source 700a is arranged to be distributed as widely as possible in the spaced space between the first column block 100a and the second column block 200a, and to emit the same light as much as possible.
  • the light source 700a may be operably connected to a lens (not shown) for collecting light emitted from the light source 700a and an optical filter (not shown) for filtering light of a specific wavelength band.
  • the step of detecting in real time the degree of nucleic acid amplification in the PCR chip 10 is based on the following steps. After completion of the first denaturation step of the PCR, the connection member 520a of the driving means 500a is controlled to move the PCR chip 10 from above the first row block 100a to the second row block 200a. After moving up or after the first annealing and extending (or amplifying) step of the PCR, the connecting member 520a of the driving means 500a is controlled to move the PCR chip 10 to the second row block 200a.
  • the chip holder 300a on which the PCR chip 10 is mounted is controlled to control the connecting member 520a of the driving means 500a.
  • a step of stopping on the spaced space between the row block 100a and the second row block 200a is performed.
  • light is emitted from the light source 700a, and the emitted light passes through the light transmissive PCR chip 10, specifically, the reaction channel of the PCR chip 10, in which case the reaction channel)
  • the light detector 800a detects an optical signal generated by amplification of the nucleic acid therein.
  • the light passing through the light transmissive PCR chip 10 may reach the light exit portion 800a by passing through the driving means 500a, specifically, the through portion 530a disposed on the rail 510a. have. Therefore, according to the PCR device according to the third embodiment of the present invention, by monitoring the reaction result by the amplification of nucleic acid (phosphorescent material bound) in the reaction channel in real time during each cyclic step of the PCR by The amount of target nucleic acid included in the initial reaction sample can be measured and analyzed in real time.
  • nucleic acid phosphorescent material bound
  • 4A to 4I are diagrams illustrating a PCR device according to a fifth embodiment of the present invention.
  • the PCR device includes a substrate 10b, a heating layer 20b including conductive nanoparticles disposed on the substrate 10b, an insulating protective layer 30b disposed on the heating layer, and a connection arrangement with the heating layer.
  • a PCR chip 10 according to the third embodiment of the present invention disposed to be in contact with the upper surface of the light transmissive thermal block 100b.
  • the substrate 10b is a plate of light transmissive material, and may be light transmissive glass or light transmissive plastic material.
  • the heating layer 20b serves as a heat source of the light transmitting thermal block 100b for performing the denaturation step, annealing step, and extension (or amplification) step of PCR.
  • the conductive nanoparticle may be an oxide semiconductor material or a material to which an impurity selected from the group consisting of In, Sb, Al, Ga, C, and Sn is added to the oxide semiconductor material.
  • the heat generating layer 20 may have a loose texture structure in which the conductive nanoparticles are physically linked to each other, and may generate a close-packed texture according to heat treatment conditions of a manufacturing process. It may also have a complete film state.
  • the conductive nanoparticles are present in a dispersed state in a solvent, the conductive nanoparticles can be easily stacked on the substrate 10b, so that the thickness of the heat generating layer 20b can be easily adjusted by controlling the number of stacked layers. Can be.
  • the conductivity of the heating layer 20b may be easily adjusted by adjusting the concentration of the dispersion liquid containing the conductive nanoparticles.
  • an adhesion strengthening layer (not shown) may be formed between the substrate 10b and the heating layer 20b to strongly fix the heating layer 20b to the substrate 10b.
  • the adhesion reinforcing layer may be formed of silica or a polymer, may include conductive nanoparticles may also play the same role as the heating layer.
  • the heating layer 20b may be transparent.
  • the wavelength of visible light is 400 to 700 nm, and when a heat generating layer including conductive nanoparticles is formed to have a thickness of 1/4 or less of such wavelength, for example, about 100 nm or less, light transmittance may be obtained.
  • the insulating protective layer 30b is for physically and / or electrically protecting the heating layer 20b and may include an insulating material.
  • the insulating material may be selected from the group consisting of dielectric oxides, perylenes, nanoparticles, and polymer films.
  • the insulation protection layer 30b may be transparent.
  • the electrode 40b is connected to the heat generating layer 20 directly or indirectly to supply power to the heat generating layer 20b.
  • the electrode 40b may be selected from, for example, a metal material, a conductive epoxy, a conductive paste, a solder, and a conductive film.
  • the electrodes 40b are connected to both sides of the heating layer 20b, but may be connected at various operable positions if power can be supplied to the heating layer 20b.
  • the electrode 40b may be included in the PCR device or electrically connected to an externally arranged power source.
  • the electrode 40b directly contacts the heat generating layer 20b, and connects the heat generating layer 20b to an external circuit (not shown) through a wire (not shown).
  • the terminal may be arranged to be stably fixed to the electrode 40b.
  • the light transmissive thermal block 100b includes a chip contact 50b to which a PCR chip (not shown) contacts at least a portion of an upper surface thereof.
  • the PCR chip 10 may be heated or cooled according to the heat supply or recovery of the light transmitting thermal block 100b by contacting the chip contact part 50b to perform each step of PCR.
  • the PCR chip 10 may directly or indirectly contact the chip contact 50.
  • the PCR device including the light transmitting thermal block 100b has many advantages over the conventional PCR device using a conventional heater, ceramic heater, or metal heater as a thermal block.
  • the conductive nanoparticles are used as the heat source, there is no fear of disconnection of the heating layer, and since the conductive nanoparticles are directly heated, high thermal efficiency and low power consumption can be obtained (for example, the light transmittance If the heat block is about 2X2 cm size, it can generate heat with a voltage of about 12V.) Since it is not a metal material, it hardly oxidizes and corrodes, so it has excellent durability. In addition, since the light transmittance may be obtained when the substrate 10b, the heat generating layer 20b, and the insulating protective layer 30b are manufactured, when included together with the light providing unit and the light detecting unit to be described below, the sample 10 may be included in the sample solution.
  • the light transmitting heat block 100b can be slimmed, thereby allowing the light transmitting heat to be reduced. Miniaturization of the PCR device including the block 100b is possible.
  • the conductive nanoparticles are uniformly distributed in the heat generating layer 20b, uniform heat distribution and rapid temperature control of the light transmissive thermal block 100b are possible. You can get it quickly. The uniformity of the heat distribution of the light transmitting thermal block 100b and the rapidity of temperature control can be confirmed as an experimental result according to FIG. 2.
  • FIG. 4B illustrates a temperature change with time of the light transmissive thermal block 100b included in the PCR device according to the fifth embodiment of the present invention.
  • the temperature distribution is observed by applying electric power to the calcite heater, the ceramic heater, or the metal heater used as the thermal block in the conventional PCR apparatus, and the temperature distribution by applying electric power to the light transmitting thermal block 100b through the electrode 40b.
  • the temperature distribution on the existing heater was not uniform throughout the heater surface, but the temperature distribution on the light transmissive heat block 100b was observed to be overall uniform compared to the conventional heater.
  • power was applied to the light transmitting thermal block 100b through the electrode 40b to observe a temperature change of the light transmitting thermal block 100b over time.
  • the temperature rise was shown to be up to 17 °C / sec, which indicates that the temperature rise of the typical conventional heaters (for example, Bio-Rad's CFX96) is significantly higher than the maximum rise of 5 °C / sec. Can be.
  • FIG. 4C shows a light transmissive thermal block 100b included in a PCR device according to a fifth embodiment of the present invention in which a light absorbing layer 60b is disposed in contact with a bottom surface of a substrate 10b
  • FIG. 4D shows an insulating protective layer.
  • 30b illustrates a light transmissive thermal block 100b included in a PCR device according to a fifth embodiment of the present invention in which a light reflection prevention layer 70b is disposed in contact with an upper surface thereof
  • FIG. 4e illustrates a bottom surface of the substrate 10b.
  • the light absorbing layer 60b is disposed in contact with the light absorbing layer 60b, and the light reflection preventing layer 70b for preventing light reflection due to the contact between the external air layer and the insulating protective layer 30b is in contact with the upper portion of the insulating protective layer 30b.
  • a light transmissive thermal block 100b included in a PCR device according to a fifth embodiment of the present invention is shown.
  • Real time PCR real time polymerase chain reaction
  • a fluorescent material as well as a reagent required for a PCR reaction is added to a PCR chip, and the fluorescent material emits light by light of a specific wavelength according to the generation of a PCR product, thereby inducing a measurable optical signal. Therefore, in order to accurately monitor the PCR product in real time, it is necessary to increase the sensing efficiency of the optical signal as much as possible.
  • the excitation light derived from the light source may be transmitted as it is, thereby increasing the sensing efficiency of the optical signal.
  • some of the excitation light may be reflected on the light transmissive heat block 100b or reflected after passing through the light transmissive heat block 100b to act as noise of an optical signal. Therefore, preferably, the light absorbing material may be treated on the lower surface of the light transmitting thermal block 100b to further increase the sensing efficiency.
  • the light absorbing layer 60b is disposed in contact with the lower surface of the substrate 10b, and the light absorbing layer 60b includes a light absorbing material.
  • the light absorbing material may be, for example, mica, but is not limited to a material having a property of absorbing light. Therefore, the light absorption layer 60b absorbs a part of the light derived from the light source, and the generation of reflected light acting as noise of the optical signal can be suppressed as much as possible.
  • the light reflection preventing material may be treated on the upper surface of the light transmissive thermal block 100b to further increase the sensing efficiency.
  • the light reflection prevention layer 70b is disposed in contact with the upper surface of the insulating protection layer 30b, and the light reflection prevention layer 70b is combined with the insulation protection layer 30b to provide insulation protection and light reflection. Performs a protective function and includes an antireflective material.
  • the anti-reflective material may be, for example, a fluoride such as MgF 2, an oxide such as SiO 2 or Al 2 O 3, but is not limited as long as the material has a property of preventing light reflection.
  • the light absorbing material is treated on the lower surface of the light transmitting thermal block 100b, and at the same time, the light reflection preventing material is treated on the upper surface of the light transmitting thermal block 100b to further increase the sensing efficiency.
  • the reflectance of the excitation light of conventional heaters of a general metallic material is about 20 to 80%
  • the light transmitting thermal block including the light absorbing layer 60b or the antireflective layer 70b according to FIG. 4C or 4D In the case of using 100b, the light reflectance can be reduced to within 0.2% to 4%
  • the light transmitting thermal block 100b according to the present invention includes a light absorbing layer 60b and an antireflective layer 70b according to FIG. 4E. When using the light reflectance can be reduced to 0.2% or less.
  • the PCR device includes a light providing unit 200b disposed to be operable to provide light to the PCR chip 10 disposed on the chip contact unit 50b and a PCR disposed on the chip contact unit 50b.
  • the apparatus further includes a light detector 300b operably disposed to receive light emitted from the chip 10.
  • the light providing unit 200b is a module for providing light to the PCR chip 10, and the light detecting unit 300b receives the light emitted from the PCR chip 10 in the PCR chip 10. Module for measuring the PCR reaction performed.
  • the PCR device According to the PCR device according to the fifth embodiment of the present invention, the amplification of nucleic acid (phosphorescent material bound) in the reaction channel during each cyclic step of the PCR in the PCR chip 10 by By monitoring the result of the reaction in real time, it is possible to measure and analyze in real time whether the target nucleic acid contained in the initial sample solution and the degree of amplification.
  • the light providing unit 200b and the light detecting unit 300b may be disposed above or below the light transmitting thermal block 100b, or may be disposed respectively.
  • the arrangement of the light providing unit 200b and the light detecting unit 300b may vary in consideration of the arrangement relationship with other modules for optimal implementation.
  • the light providing unit ( 200b) and the light detector 300b may be disposed on the light transmitting thermal block.
  • the light providing unit 200b may include a light emitting diode (LED) light source or a laser light source 210b and a first light filter for selecting light having a predetermined wavelength from light emitted from the light source.
  • a first optical lens 240b for collecting light emitted from the first light filter, the first optical lens 240b being arranged to spread light between the light source 210b and the first light filter 230b.
  • the light source 210b includes all light sources capable of emitting light, and includes a light emitting diode (LED) light source or a laser light source.
  • the first light filter 230b selects and emits light having a specific wavelength among incident light having various wavelength bands, and may be variously selected according to the predetermined light source 210b.
  • the first light filter 230b may pass only light in a wavelength band of 500 nm or less among the light emitted from the light source 210b.
  • the first optical lens 240b collects the incident light and increases the intensity of the emitted light.
  • the first optical lens 240b increases the intensity of light irradiated onto the PCR chip 10 through the light transmitting thermal block 100b. Can be.
  • the light providing unit 200b further includes a first aspherical lens 220b disposed to spread light between the light source 210b and the first light filter 230b. By adjusting the arrangement direction of the first aspherical lens 220b, the light range emitted from the light source 210b is extended to reach the measurable area.
  • the light detector 300b includes a second optical lens 310b for collecting light emitted from the PCR chip 10 disposed on the chip contact 50b, and the light emitted from the second optical lens.
  • a second light filter 320b for selecting light having a predetermined wavelength at and an optical analyzer 350b for detecting an optical signal from the light emitted from the second light filter 320b, wherein the second light filter
  • the second aspherical lens 330b disposed between the filter 320b and the optical analyzer 350b to integrate light emitted from the second light filter 320b, wherein the second aspherical lens 330b is provided.
  • optical diode integrated device disposed between the optical analyzer 350b to remove noise of light emitted from the second aspherical lens 330b and to amplify the light emitted from the second aspherical lens 330b.
  • PDIC photodiode integrated circuit
  • the second light filter 320b selects and emits light having a specific wavelength among incident light having various wavelength bands, and is determined according to a wavelength of predetermined light emitted from the PCR chip 10 through the light transmitting thermal block 100b. Various choices can be made. For example, the second light filter 320b may pass only light in a wavelength band of 500 nm or less among predetermined light emitted from the PCR chip 10 through the light transmitting thermal block 100b.
  • the optical analyzer 350b is a module that detects an optical signal from light emitted from the second optical filter 320b. The optical analyzer 350b converts light fluorescence expressed from a sample solution into an electrical signal to enable qualitative and quantitative measurement. .
  • the light detector 300b may include a second aspheric lens 330b disposed between the second light filter 320b and the light analyzer 350b to integrate light emitted from the second light filter 320b. It may further include. By adjusting the arrangement direction of the second aspherical lens 330b, the light range emitted from the second light filter 320b is extended to reach the measurable area. In addition, the light detector 300b removes noise of light emitted from the second aspherical lens 330b between the second aspherical lens 330b and the optical analyzer 350b, and the second aspherical surface.
  • the device may further include a photodiode integrated circuit (PDIC) 340b disposed to amplify the light emitted from the lens 330b.
  • PDIC photodiode integrated circuit
  • the PCR apparatus may adjust one or more directions of light so that light emitted from the light providing unit 200b may reach the light detecting unit 300b and separate one or more light having a predetermined wavelength. It further includes dichroic filters 400x and 400y.
  • the dichroic filters 400x and 400y are modules that reflect light at an angle selectively transmitted or selectively adjusted according to the wavelength.
  • the dichroic filter 400x is disposed to be inclined at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light providing unit 200b, and selectively transmits the short wavelength component according to its wavelength and transmits the long wavelength component.
  • the dichroic filter 400y is disposed to be inclined at an angle of about 45 degrees with respect to the optical axis of the light reflected from the PCR chip 10 and the light transmitting thermal block 100b, and the light is selectively shortened according to its wavelength.
  • the long wavelength component is reflected at right angles to reach the light detector 300b.
  • the light reaching the light detector 300b is converted into an electrical signal in the optical analyzer to indicate whether the nucleic acid is amplified and the degree of amplification.
  • 5A to 5D are diagrams of a PCR device according to a sixth embodiment of the present invention.
  • PCR apparatus according to a sixth embodiment of the present invention according to Figures 5a to 5d is provided with a heater group having at least one heater, at least two heater groups and the two or more heater groups are spaced apart so that mutual heat exchange does not occur Two or more heater units, the thermal block including a contact surface of a PCR chip to accommodate a target sample on at least one surface; An electrode unit having an electrode connected to supply electric power to heaters provided in the thermal block; And a PCR chip 10 according to a third embodiment of the present invention disposed to be in contact with the heat block to allow heat exchange with one or more heaters provided in the heat block.
  • the thermal block 100c is a module implemented to supply heat to a target sample at a specific temperature to perform a PCR.
  • the thermal block 100c includes a contact surface of a PCR chip 10 on which at least one surface a target sample is accommodated. In contact with one side of the chip 10, PCR is performed by supplying heat to a target sample present in one or more reaction channels.
  • the thermal block 100c is based on a substrate.
  • the substrate may be made of any material such that physical and / or chemical properties thereof do not change due to heating and temperature maintenance of a heater disposed in the substrate, and mutual heat exchange does not occur between two or more heaters spaced apart in the substrate. Can be.
  • the substrate may be made of plastic, glass, silicon, or the like, and may be implemented transparently or semitransparently.
  • the thermal block 100c may have a planar shape as a whole, but is not limited thereto.
  • the heat block 100c includes a heater group including one or more heaters, two or more heater groups, and the two or more heater groups are repeatedly arranged at least two heater units spaced apart from each other so that mutual heat exchange does not occur.
  • the contact surface of the PCR chip 10 may be implemented on at least one surface of the thermal block 100c and may be implemented in various shapes for efficiently supplying heat to the PCR chip 10 in which a target sample is accommodated.
  • a planar shape or a pillar shape is preferable so that the surface area of a contact surface is wide.
  • the heaters 111c, 112c, 121c, 122c, 131c, and 132c are heat generating elements, and heat wires (not shown) may be disposed therein.
  • the heating wire may be operably connected with various heat sources to maintain a constant temperature, and may be operably connected with various temperature sensors for monitoring the temperature of the heating wire.
  • the heating wire may be arranged to be symmetrical in the vertical direction and / or the horizontal direction with respect to the surface center point of the heater in order to maintain the internal temperature of the heater as a whole.
  • the heater may have a thin film heater (not shown) disposed therein.
  • the thin film heater may be disposed at regular intervals in the vertical direction and / or the left and right directions with respect to the center point of the heater surface in order to maintain the internal temperature of the heater as a whole.
  • the heater is a heating element, and may itself be a metal material, for example, chromium, aluminum, copper, iron, silver, and the like, for even heat distribution and rapid heat transfer over the same area.
  • the heater is a light-transmitting heating element, for example, conductive nanoparticles including an oxide semiconductor material or a material added with impurities selected from the group consisting of In, Sb, Al, Ga, C and Sn to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive polymeric materials, carbon nanotubes, and graphene.
  • the heater is preferably a light transmitting heating element.
  • the heater groups 110c, 120c, and 130c are units including one or more heaters, and are regions that maintain a temperature for performing a denaturation step, annealing step, and / or an extension step for PCR.
  • Two or more heater groups are disposed in the heat block 100c, and the two or more heater groups are spaced apart from each other so that mutual heat exchange does not occur.
  • Two to four heater groups may be included in the thermal block 100c. That is, the thermal block includes two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature.
  • the thermal block includes three heater groups, wherein the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group has the PCR extension step temperature.
  • the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater The group may maintain the PCR extension step temperature, the second heater group may maintain the PCR denaturation step temperature, and the third heater group may maintain the PCR annealing step temperature.
  • the heater group may be disposed three times in the thermal block 100c to maintain three temperatures for performing PCR, that is, a temperature for performing a denaturation step, an annealing step, and an extension step, and more preferably, The heater group may be disposed twice in the thermal block 100c to maintain two temperatures for performing PCR, that is, denaturation and annealing / extension, respectively, but are not limited thereto.
  • the heater group is disposed in the heat block 100c twice, and when performing the two steps for performing PCR, that is, the denaturation step and the annealing / extension step, three steps for performing the PCR, i.
  • the temperature for performing the denaturation step is 85 °C to 105 °C, preferably 95 °C
  • the temperature for performing the annealing step is 40 °C to 60 °C, preferably 50 °C
  • the temperature for performing the extension step is 50 °C to 80 °C, preferably 72 °C
  • the temperature for performing the denaturation step is 85 °C to 105 °C, preferably 95 ° C.
  • the temperature for performing the annealing / extension step is 50 ° C.
  • the heater group may further include a heater that serves as a temperature buffer.
  • the heater units 10c and 20c are units including the two or more heater groups including the one or more heaters, and an area in which one cycle including a denaturation step, annealing step, and / or extension step for performing PCR is completed. to be.
  • the heater unit is repeatedly arranged at least two in the heat block 100c.
  • the heater unit may be repeatedly arranged in the heat block 100c 10 times, 20 times, 30 times, or 40 times, but is not limited thereto.
  • the heat block 100c includes heater units 10c and 20c that are repeatedly arranged, two heater groups 110c and 120c included therein, and one heater 111c and 121c respectively included therein.
  • a two-step temperature for performing the PCR that is, one temperature of the denaturation step and one temperature of the annealing / extension step are sequentially provided.
  • the first heater 111c maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C. such that the first heater group 110c provides a temperature for performing the modification step.
  • the second heater 121c maintains one temperature in the range of 50 ° C. to 80 ° C., preferably 72 ° C. such that the second heater group 120 c provides a temperature for performing the annealing / extension step.
  • 100c) sequentially and repeatedly provides a two-step temperature for performing PCR in the first heater unit 10c and the second heater unit 20c.
  • the heat block 100c includes heater units 10c and 20c that are repeatedly arranged, two heater groups 110c and 120c included therein, and two heaters 111c and 112c respectively included therein.
  • 121c, 122c) provides two steps of temperature for performing PCR, that is, two temperatures of the denaturation step and two temperatures of the annealing / extension step.
  • the first heater 111c has one temperature in the range of 85 ° C to 105 ° C
  • the second heater 112c has one temperature that is the same as or different from the temperature of the first heater 111c in the range of 85 ° C to 105 ° C.
  • the third heater 121c is one temperature in the range of 50 °C to 80 °C
  • the fourth heater 122c is 50 °C to
  • the thermal block 100c may be maintained by maintaining a temperature equal to or different from the temperature of the third heater 121c in the range of 80 ° C. such that the second heater group 120c provides a temperature for performing an annealing / extension step. Provides sequentially two-step temperature for performing PCR in the first heater unit 10c and the second heater unit 20c.
  • the heat block 100c includes heater units 10c and 20c that are repeatedly arranged, three heater groups 110c, 120c and 130c respectively included therein, and one heater 111c respectively included therein.
  • 121c and 131c) sequentially provide three step temperatures for performing PCR, that is, one temperature of the denaturation step, one temperature of the annealing step, and one temperature of the extension step.
  • the first heater 111c maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C. such that the first heater group 110c provides a temperature for performing the modification step.
  • the second heater 121c maintains one temperature, preferably 50 ° C, in the range of 40 ° C to 60 ° C so that the second heater group 120c provides a temperature for performing the annealing step, and the third heater 131c Is maintained at a temperature in the range of 50 ° C to 80 ° C, preferably 72 ° C, so that the third heater group 130c provides a temperature for performing the extension step, whereby the thermal block 100c is configured as a first heater unit.
  • step 10c and the second heater unit 20c three steps of temperature for performing PCR are repeatedly provided sequentially.
  • heater units 10c and 20c that are repeatedly arranged, three heater groups 110c, 120c and 130c included therein, and two heaters 111c, 112c, 121c, 122c and 131c respectively included therein are included.
  • 132c provides three steps of temperature for performing PCR, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step.
  • the first heater 111c has one temperature in the range of 85 ° C to 105 ° C
  • the second heater 112c has one temperature that is the same as or different from the temperature of the first heater 111c in the range of 85 ° C to 105 ° C.
  • the third heater 121c is one temperature in the range of 40 °C to 60 °C
  • the fourth heater 122c is 40 °C to
  • the second heater group 120c provides a temperature for performing the annealing step by maintaining one temperature equal to or different from the temperature of the third heater 121c in the range of 60 ° C
  • the fifth heater 131c is 50 ° C.
  • the sixth heater 132c in the range of 1 to 80 ° C. maintains one temperature that is the same or different from the temperature of the fifth heater 131c in the range of 50 ° C. to 80 ° C. so that the third heater group 130c extends.
  • the thermal block 100c performs PCR in the first heater unit 10c and the second heater unit 20c.
  • a third step for temperature provides successively repeated.
  • the rate of change of temperature can be greatly improved by repeatedly disposing two or more heaters maintaining a constant temperature.
  • the temperature change rate is within the range of 3 ° C to 7 ° C per second
  • the rate of change of temperature between the heaters is within the range of 20 °C to 40 °C per second can greatly shorten the reaction time.
  • the heaters are spaced apart so that mutual heat exchange does not occur, and as a result, in the nucleic acid amplification reaction that can be greatly affected by minute temperature changes, the denaturation step, annealing step and extension step (or the denaturation step and annealing).
  • the heater unit may be repeatedly arranged ten times. That is, the heater unit may be repeatedly arranged in 10 times, 20 times, 30 times, 40 times, 50 times, etc. in consideration of the PCR circulation cycle according to the type of the user or target sample to be PCR, which is particularly limited.
  • the heater unit may be repeatedly arranged in half of the predetermined PCR cycle.
  • the heater unit may be repeatedly arranged ten times.
  • the target sample solution is repeated 10 times the PCR cycle from the inlet 304c to the outlet 305c in the reaction channel 303c disposed in the nucleic acid amplification reaction unit 300c to be described in detail below.
  • the PCR cycle may be repeated 10 times from the outlet portion 305c toward the inlet portion 304c.
  • the electrode unit is a module that receives power from a power supply unit (not shown) and supplies power to heat the heat block 100c.
  • the electrode is connected to supply an electric power to the heaters provided in the heat block 100c. It may include.
  • the first electrode of the thermal block 100c is connected to supply power to the first heater 110c, and the second electrode is connected to supply power to the second heater 120c, but is not limited thereto. Instead, the electrode unit may be disposed to be driven outside the thermal block 100c.
  • the first heater 110c maintains the PCR denaturation step temperature, for example 85 °C to 105 °C and the second heater 120c maintains the PCR annealing / extension stage temperature, for example 50 °C to 80 °C
  • the first electrode may receive power for maintaining the PCR denaturation step temperature from the power supply
  • the second electrode may receive power for maintaining the PCR annealing / extension step temperature from the power supply.
  • the first electrode and the second electrode may be connected to the first heater 110c and the two or more second heaters 120c that are repeatedly disposed in the thermal block 100c.
  • the first electrode and the second electrode may be a conductive material such as gold, silver, copper, and the like, and are not particularly limited.
  • the PCR chip 10 may be supplied with heat from the first heater 110c and the second heater 120c in contact with the heat block 100c to allow heat exchange with the heat block 100c. have.
  • the PCR chip 10 is one or more reactions extending to extend in the longitudinal direction through the upper corresponding portion 301c of the first heater (110c) and the upper corresponding portion (302c) of the second heater (120c) Channel 14 may be provided.
  • the reaction channel 14 passes in the longitudinal direction by connecting the upper corresponding portion of the first heater 110c disposed in the thermal block 100c and the upper corresponding portion of the second heater 120c in fluid communication. Is extended.
  • the upper corresponding portion of the first heater 110c of the reaction channel 14 is a region where PCR denaturation reaction of nucleic acid present in the target sample occurs, and the upper corresponding portion of the second heater 120c is present in the target sample. It may be a region where PCR annealing / extension reaction of the nucleic acid occurs. That is, PCR may be performed while the target sample flowing through the reaction channel 14 passes through the upper corresponding portion of the first heater 110c and the upper corresponding portion of the second heater 120c in succession.
  • the PCR chip 10 may include an inlet 15 and an outlet 16 at both ends of the one or more reaction channels 14, respectively.
  • the one or more reaction channels 14 extend so as to pass through the upper corresponding portion of the first heater 110c disposed best of the heater unit and the upper corresponding portion of the second heater 120c disposed last in a straight length direction. It is preferable to arrange. Therefore, the target sample introduced through the inlet part 15 passes through the reaction channel 14 in the longitudinal direction, and is respectively modified with PCR in the upper corresponding part of the first heater 110c and the upper corresponding part of the second heater. Repeating the step and PCR annealing / extension step may be discharged to the outside through the outlet.
  • the PCR chip 10 may be a planar shape as a whole, but is not limited thereto.
  • the PCR chip 10 may be implemented with a light transmissive material, if the PCR device according to the fifth embodiment of the present invention is used for real-time (real-time PCR) the PCR chip 10 is optical It is preferred to be implemented with a transparent material.
  • Foot and mouth virus A (Genbank ID number: NC011450), foot and mouth virus O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 from Genbank (Genbank ID number: NC011451), and the genetic information on the foot-and-mouth virus SAT 2 (Genbank ID number: NC011452) was obtained, and the base sequence of each serotype was commissioned by Cosmogenetech Co., Ltd. to proceed with gene synthesis, Vector pUC57 The synthesized gene was inserted into and cloned. In addition, the accuracy of the inserted gene was confirmed by sequencing.
  • Transformation was performed using DH5 ⁇ (bacteria) to plasmid DNA by selecting and culturing only colonies that were cloned correctly after plating on culture plates.
  • the sequencing of the extracted plasmid DNA was performed to analyze the nucleotide sequence of the plasmid DNA cloned (cloning) and confirmed that it was amplified according to the gene sequence.
  • PCR sample solution conditions of Table 2 are for use in other companies' PCR device (Bio-Rad: CFX-connect, below), and PCR sample solution conditions of Table 3 is applied to the PCR device according to the fourth embodiment of the present invention. It is to use.
  • PCR was performed using the PCR apparatus according to the fourth embodiment of the present invention and the third-party PCR apparatus according to the PCR conditions of Tables 4 and 5 below.
  • the PCR conditions of Table 4 are applied to the PCR device of the other company, the PCR conditions of Table 5 is applied to the PCR device according to the fourth embodiment of the present invention.
  • FIG. 6 is a result of PCR comparison with the foot-and-mouth virus A plasmid sample
  • FIG. 7 is a result of PCR comparison with the foot-and-mouth virus O plasmid sample
  • FIG. 8 shows a foot-and-mouth virus C plasmid sample.
  • 9 is a result of PCR comparison experiment for the foot-and-mouth virus virus plasmid sample
  • FIG. 10 is a result of PCR comparison for the foot-and-mouth virus SAT 1 plasmid sample
  • FIG. 11 is a foot-and-mouth virus SAT It is the result of PCR comparison experiment with 2 plasmid samples.
  • FIGS. 6 to 11 FIGS.
  • a to b each show a Ct value of each foot-and-mouth disease virus plasmid sample concentration compared to a negative control group using a third-party PCR device and a PCR device according to a fourth embodiment of the present invention.
  • Tables c to d show changes in cycles (x-axis) fluorescence (y-axis) of PCR amplification products by a third-party PCR device and a PCR device according to a fourth embodiment of the present invention, respectively.
  • Figures e to f are electrophoresis pictures of the PCR amplification products by the third-party PCR device and the PCR device according to the fourth embodiment of the present invention, respectively.

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Abstract

The present invention relates to a device for detecting foot and mouth disease (FMD) according to a serum type, and a method for detecting an foot and mouth disease outbreak by using same, and more specifically, the device for detecting food and mouth disease according to the serum type is for detecting at least one gene selected from a group consisting of: foot and mouth disease virus A (Genbank ID number: NC011450); foot and mouth disease virus O (Genbank ID number: NC004004); foot and mouth disease virus C (Genbank ID number: NC002554), foot and mouth disease virus Asia (Genbank ID number: NC004915); foot and mouth disease SAT 1 (Genbank ID number: NC011451); and foot and mouth disease SAT 2 (Genbank ID number: NC011452). According to the present invention, a foot and mouth disease outbreak can be accurately and rapidly confirmed at a low cost, thereby significantly contributing to the prevention of and rapid response to the spreading of foot and mouth disease.

Description

혈청 타입별 구제역 검출용 프라이머 세트, 이를 이용한 PCR 장치, 및 이를 이용한 구제역 검출 방법Set of primers for detecting foot-and-mouth disease by serum type, PCR device using the same, and foot-and-mouth detection method using the same
본 발명은 구제역(Foot and mouth disease, FMD) 발병 여부를 검출하기 위한 검출 장치 및 검출 방법에 관한 것이다.The present invention relates to a detection device and a detection method for detecting whether foot and mouth disease (FMD).
구제역은 발굽이 둘로 갈라진 동물, 예를 들어 소, 돼지 등에 감염되는 바이러스 수포성 질병으로 빠른 복제와 전파력을 특징으로 한다. 구제역은 전형적인 가축 질병이기 때문에 그 경제적 중요성으로 인해 국제수역사무국(OIE)에 의하여 관리되고 있고, 구제역 발병시 가축 동물의 국가 간 수출입에 있어서 큰 제한이 뒤따른다. 구제역에 감염된 가축은 고열이 발생하지만 이틀에서 사흘이 지나면 열이 가라앉고, 입 속에 생기는 수포로 인해 거품이 많고 끈적끈적한 침을 심하게 흘리며, 발굽에도 수포가 생겨 터지기도 하며 걸음을 절뚝거리기도 한다. 또한, 다 자란 개체의 경우 체중 감소를 겪기도 하며 이런 체중 감소는 몇 달 동안 회복되지 않고, 수컷의 경우 고환이 부풀기도 하며, 젖소의 우유 생산량이 급격히 감소할 수 있다. 구제역 발병이 시작되면 급속하게 확산되기 때문에 축산 업계뿐만 아니라 국가 경제에도 심각한 손해를 입힐 수 있다. 따라서, 이러한 피해를 최소화하고, 초기에 신속한 예방 및 처리를 하기 위해서라도 구제역 발병 여부를 정확하고 신속하며 경제적으로 확인할 수 있는 방법이 절실히 요구된다. Foot-and-mouth disease is a viral bullous disease that infects animals with two hoofs, such as cattle and pigs, and is characterized by rapid replication and transmission. Because foot-and-mouth disease is a typical livestock disease, its economic importance is managed by the International Water Services Bureau (OIE), and large outbreaks in cross-border imports and exports of livestock animals occur when foot-and-mouth disease develops. Animals infected with foot-and-mouth disease develop a high fever, but after two days, the heat subsides, the blisters in the mouth cause a lot of foamy and sticky saliva, and the hooves develop blisters and limping. In addition, mature individuals may experience weight loss, and such weight loss may not recover for months, swelling of the testicles in males, and the milk yield of cows may drop dramatically. Since the onset of foot and mouth disease spreads rapidly, it can seriously damage the national economy as well as the livestock industry. Therefore, there is an urgent need for a method that can accurately and promptly and economically determine whether foot-and-mouth disease occurs even in order to minimize such damage and to promptly prevent and treat the disease early on.
한편, 실시간 PCR(Real-time Polymerase Chain Reaction)은 PCR 증폭 산물의 증가를 PCR의 매 주기마다 실시간으로 관찰하는 방법으로서, PCR 증폭 산물과 반응하는 형광 물질의 검출과 정량으로 해석하는 방법이다. 실시간 PCR은 기존의 PCR이 최종 단계를 마치고 겔 상에서 염색하여 전기영동 후 PCR 증폭 산물을 확인하는 것에 비해, 전기영동의 추가 작업이 필요 없고, 정확도 및 민감도가 뛰어나며, 재현율이 높고, 자동화가 가능하며, 결과를 수치화할 수 있고, 신속하고 간편하며, EtBr(Ethidium Bromide)과 같은 염색제에 의한 오염 및 자외선 조사 등의 유해 문제에 따른 생물학적 안전성이 뛰어나고, 자동으로 특이 유전자의 증폭 유무를 확인할 수 있다는 장점이 있다. 따라서, 실시간 PCR을 통해 PCR 또는 항원/항체와 같은 정성적인 결과가 아닌 높은 특이도를 갖는 정량적인 결과를 확인할 수 있다. 또한 형광 표지 인자로 표지된 프로브를 이용하기 때문에 DNA 칩이나 항원/항체 반응에 사용되는 시료의 양보다 적은 양의 시료로도 결과를 확인할 수 있다.On the other hand, real-time PCR (Real-time Polymerase Chain Reaction) is a method for observing the increase of the PCR amplification product in real time every cycle of the PCR, it is a method for interpreting by the detection and quantification of the fluorescent material reacted with the PCR amplification product. Real-time PCR does not require any additional electrophoresis, it is highly accurate, sensitive, has high reproducibility, and is automated, compared to conventional PCR, which is stained on gel after final step to identify PCR amplification products after electrophoresis. It is possible to quantify the results, and it is quick and easy, and has excellent biological safety due to harmful problems such as contamination by dyes such as EtBr (Ethidium Bromide) and UV irradiation, and can automatically check whether a specific gene is amplified. There is this. Thus, real-time PCR can confirm quantitative results with high specificity rather than qualitative results such as PCR or antigen / antibody. In addition, since probes labeled with fluorescent labeling factors are used, the results can be confirmed with a sample smaller than the amount of a sample used for DNA chip or antigen / antibody reaction.
따라서, 대상 시료 내의 구제역 발병 여부를 신속하고 정확하게 진단하기 위해 실시간 PCR 방법을 이용한 구제역 검출 장치 및 이를 이용하는 구제역 검출 방법의 필요성이 요구되고 있는 실정이다.Therefore, there is a need for an apparatus for detecting foot-and-mouth disease using a real-time PCR method and a foot-and-mouth disease detection method using the same in order to quickly and accurately diagnose foot-and-mouth disease in a target sample.
본 발명의 일 실시예들은 구제역의 혈청 타입별 원인 바이러스에 관한 유전자를 동시에 특이적으로 증폭할 수 있는 프라이머 세트, 일련의 구제역 검출 장치 및 검출 방법을 제공하고자 한다.One embodiment of the present invention is to provide a primer set, a series of foot-and-mouth disease detection device and a detection method capable of simultaneously specifically amplifying genes related to the cause virus of each type of foot-and-mouth disease.
본 발명의 제1 실시예는 서열번호 1의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머 및 서열번호 2의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머로 이루어진, 구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)로 구성된 군으로부터 1 이상 선택된 유전자를 검출하기 위한, 혈청 타입별 구제역(Foot and mouth disease, FMD) 검출용 프라이머 세트를 제공한다.The first embodiment of the present invention comprises foot-and-mouth virus A, which comprises a primer comprising 15 or more consecutive nucleotides of SEQ ID NO: 1 and a primer comprising 15 or more consecutive nucleotides of SEQ ID NO: Genbank ID number: NC011450), foot and mouth virus O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451 ), And foot and mouth virus SAT 2 (Genbank ID number: NC011452) provides a primer set for detecting foot and mouth disease (Foot and mouth disease (FMD)) for detecting one or more genes selected from the group consisting of.
본 발명의 제2 실시예는 서열번호 3의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머 및 서열번호 4의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머로 이루어진, 구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)로 구성된 군으로부터 1 이상 선택된 유전자를 검출하기 위한, 혈청 타입별 구제역(Foot and mouth disease, FMD) 검출용 프라이머 세트를 제공한다.A second embodiment of the present invention comprises foot-and-mouth virus A, which comprises a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 3 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 4 Genbank ID number: NC011450), foot and mouth virus O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451 ), And foot and mouth virus SAT 2 (Genbank ID number: NC011452) provides a primer set for detecting foot and mouth disease (Foot and mouth disease (FMD)) for detecting one or more genes selected from the group consisting of.
본 발명의 제3 실시예는 제1 판; 상기 제1 판 상에 배치되고, 1 이상의 반응 채널을 구비하는 제2 판; 및 상기 제2 판 상에 배치되고, 상기 1 이상의 반응 채널의 양 말단과 연결되되 개폐 가능하도록 구현된 유입부 및 유출부를 구비하는 제3 판을 포함하는 것으로서, 상기 1 이상의 반응 채널 내에 본 발명의 제1 실시예에 따른 프라이머 세트를 포함하는 것을 특징으로 하는 PCR(Polymerase Chain Reaction) 칩을 제공한다. 본 발명의 제3 실시예에 있어서, 상기 제1 판 및 제3 판은 폴리디메틸실옥산(polydimethylsiloxane, PDMS), 사이클로올레핀코폴리머(cycle olefin copolymer, COC), 폴리메틸메타크릴레이트(polymethylmetharcylate, PMMA), 폴리카보네이트(polycarbonate, PC), 폴리프로필렌카보네이트(polypropylene carbonate, PPC), 폴리에테르설폰(polyether sulfone, PES), 및 폴리에틸렌텔레프탈레이트(polyethylene terephthalate, PET), 및 그의 조합물로 구성된 군으로부터 선택되는 재질을 포함하고, 상기 제2 판은 폴리메틸메타크릴레이트(polymethylmethacrylate, PMMA), 폴리카보네이트(polycarbonate, PC), 사이클로올레핀 코폴리머(cycloolefin copolymer, COC), 폴리아미드(polyamide, PA), 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리페닐렌 에테르(polyphenylene ether, PPE), 폴리스티렌(polystyrene, PS), 폴리옥시메틸렌(polyoxymethylene, POM), 폴리에테르에테르케톤(polyetheretherketone, PEEK), 폴리테트라프로오르에틸렌(polytetrafluoroethylene, PTFE), 폴리비닐클로라이드(polyvinylchloride, PVC), 폴리비닐리덴 플로라이드(polyvinylidene fluoride, PVDF), 폴리부틸렌테레프탈레이트(polybutyleneterephthalate, PBT), 불소화에틸렌프로필렌(fluorinated ethylenepropylene, FEP), 퍼플로로알콕시알칸(perfluoralkoxyalkane, PFA), 및 그의 조합물로 구성된 군으로부터 선택되는 열 가소성 수지 또는 열 경화성 수지 재질을 포함할 수 있다. 또한, 상기 PCR 칩은 플라스틱 재질로 구현되되, 광 투과성을 갖도록 구현될 수 있다. 또한, 상기 PCR 칩은 상기 1 이상의 반응 채널 내에 dATP, dCTP, dGTP, 및 dTTP를 포함하는 혼합물, DNA 중합효소 및 검출 가능한 표지를 더 포함할 수 있다. 또한, 상기 검출 가능한 표지는 Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 및 티아졸 오렌지(thiazole orange)로 이루어진 군으로부터 선택될 수 있다.A third embodiment of the present invention is the first plate; A second plate disposed on the first plate and having one or more reaction channels; And a third plate disposed on the second plate, the third plate having an inlet and an outlet connected to both ends of the at least one reaction channel and configured to be openable and closed. It provides a PCR (Polymerase Chain Reaction) chip comprising a primer set according to the first embodiment. In a third embodiment of the present invention, the first plate and the third plate is polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate, PMMA) ), Polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof The second plate is made of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (CCO), polyamide (PA), polyethylene (polyethylene, PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) ), Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate ( polybutyleneterephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), and combinations thereof, and may comprise a thermoplastic or thermosetting resin material selected from the group consisting of. In addition, the PCR chip is implemented in a plastic material, it may be implemented to have a light transmission. In addition, the PCR chip may further comprise a mixture comprising dATP, dCTP, dGTP, and dTTP, DNA polymerase and detectable label in the one or more reaction channels. In addition, the detectable label is Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680 , Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green , SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 and thiazole orange.
본 발명의 제4 실시예는 기판 상에 배치된 제1 열 블록; 상기 기판 상에 상기 제1 열 블록과 이격 배치된 제2 열 블록; 및 상기 제1 열 블록 및 제2 열 블록 위로 구동 수단에 의해 좌우 및/또는 상하 이동 가능하고, 본 발명의 제3 실시예에 따른 PCR 칩이 장착된 칩 홀더를 포함하는 PCR 장치를 제공한다. 본 발명의 제3 실시예에 있어서, 상기 제1 열 블록과 제2 열 블록 사이에 광원이 더 배치되고, 상기 칩 홀더 위에 상기 광원으로부터 방출되는 광을 검출하기 위한 광 검출부가 더 배치되거나, 또는 상기 제1 열 블록과 제2 열 블록 사이에 광원으로부터 방출되는 광을 검출하기 위한 광 검출부가 더 배치되고, 상기 칩 홀더 위에 광원이 더 배치될 수 있다.A fourth embodiment of the present invention includes a first thermal block disposed on a substrate; A second thermal block spaced apart from the first thermal block on the substrate; And a chip holder movable left, right and / or up and down by the driving means over the first row block and the second row block, and equipped with a PCR chip according to the third embodiment of the present invention. In a third embodiment of the present invention, a light source is further disposed between the first column block and the second column block, and a light detector for detecting light emitted from the light source is further disposed on the chip holder, or A light detector for detecting light emitted from the light source may be further disposed between the first and second heat blocks, and a light source may be further disposed on the chip holder.
본 발명의 제5 실시예는 기판, 상기 기판 상에 배치된 도전성 나노 입자를 포함하는 발열층, 상기 발열층 상에 배치된 절연 보호층 및 상기 발열층과 연결 배치된 전극을 구비하되, 광투과성을 갖도록 구현된 광투과성 열 블록; 및 상기 광투과성 열 블록의 상부 면에 접촉 가능하도록 배치된, 본 발명의 제3 실시예에 따른 PCR 칩을 포함하는 PCR 장치를 제공한다. 본 발명의 제4 실시예에 있어서, 상기 기판은 광 투과성 유리 또는 플라스틱 재질이고, 상기 발열층에 포함된 도전성 나노 입자는 산화물 반도체 물질 또는 상기 산화물 반도체 물질에 In, Sb, Al, Ga, C 및 Sn로 구성된 군으로부터 선택된 불순물이 첨가된 물질이고, 상기 절연 보호층은 유전체 산화물, 페릴린, 나노 입자 및 고분자 필름으로 구성된 군으로부터 선택되는 것이고, 상기 전극은 금속 물질, 전도성 에폭시, 전도성 페이스트, 솔더 및 전도성 필름으로 구성된 군으로부터 선택될 수 있다. 또한, 상기 광투과성 열 블록의 기판의 하부 면은 흡광 물질이 포함된 흡광층이 접촉 배치되거나, 또는 상기 광투과성 열 블록의 절연 보호층의 상부 면은 광반사 방지 물질이 포함된 광반사방지층이 접촉 배치될 수 있다. 또한, 상기 PCR 장치는 상기 칩 접촉부에 배치되는 PCR 칩에 광을 제공하도록 구동가능하게 배치된 광 제공부 및 상기 칩 접촉부에 배치되는 PCR 칩으로부터 방출되는 광을 수용하도록 구동가능하게 배치된 광 검출부를 더 포함할 수 있다. A fifth embodiment of the present invention includes a substrate, a heat generating layer including conductive nanoparticles disposed on the substrate, an insulating protective layer disposed on the heat generating layer, and an electrode disposed in connection with the heat generating layer, but is light-transparent A light transmissive thermal block implemented to have; And it provides a PCR device comprising a PCR chip according to a third embodiment of the present invention, arranged to be in contact with the upper surface of the light transmitting thermal block. In the fourth embodiment of the present invention, the substrate is a light-transmissive glass or plastic material, the conductive nanoparticles included in the heat generating layer is an oxide semiconductor material or In, Sb, Al, Ga, C and It is a material to which impurities selected from the group consisting of Sn is added, the insulating protective layer is selected from the group consisting of dielectric oxide, perylene, nanoparticles and polymer film, the electrode is a metal material, conductive epoxy, conductive paste, solder And it may be selected from the group consisting of a conductive film. In addition, the lower surface of the substrate of the light-transmissive heat block is disposed in contact with the light absorbing layer containing the light absorbing material, or the upper surface of the insulating protective layer of the light-transmissive heat block is a light reflection prevention layer containing the anti-reflective material Can be placed in contact. The PCR device may further include a light providing unit operably arranged to provide light to a PCR chip disposed in the chip contact unit, and a light detection unit operatively arranged to receive light emitted from the PCR chip disposed in the chip contact unit. It may further include.
본 발명의 제6 실시예는 1 이상의 히터를 구비하는 히터 군, 상기 히터 군을 2 이상 구비하고 상기 2 이상의 히터 군은 상호 열 교환이 일어나지 않도록 이격 배치된 히터 유닛이 2 이상 반복 배치된 것으로서, 적어도 일 면에 표적 샘플이 수용되는 PCR 칩의 접촉 면을 구비하는 열 블록; 상기 열 블록에 구비된 히터들에 전력을 공급하도록 연결된 전극을 구비하는 전극부; 및 상기 열 블록에 구비된 1 이상의 히터들과 열 교환이 가능하도록 상기 열 블록 상에 접촉가능하도록 배치된, 본 발명의 제3 실시예에 따른 PCR 칩을 포함하는 PCR 장치를 제공한다. 본 발명의 제5 실시예에 있어서, 상기 열 블록은 2개 내지 4개의 히터 군을 구비할 수 있다. 또한, 상기 열 블록은 2개의 히터 군을 구비하고, 상기 제1 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제2 히터 군은 PCR 어닐링/연장 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 어닐링/연장 단계 온도를 유지하고 상기 제2 히터 군은 PCR 변성 단계 온도를 유지할 수 있다. 또한, 상기 열 블록은 3개의 히터 군을 구비하고, 상기 제1 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제2 히터 군은 PCR 어닐링 단계 온도를 유지하고 상기 제3 히터 군은 PCR 연장 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 어닐링 단계 온도를 유지하고 상기 제2 히터 군은 PCR 연장 단계 온도를 유지하고 상기 제3 히터 군은 PCR 변성 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 연장 단계 온도를 유지하고 상기 제2 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제3 히터 군은 PCR 어닐링 단계 온도를 유지할 수 있다. 또한, 상기 열 블록은 광 투과성을 갖도록 구현될 수 있다. 또한, 상기 열 블록에 구비된 히터는 광 투과성 발열소자를 포함할 수 있다. 또한, 상기 전극부에 전력을 공급하기 위한 전력 공급부 및 상기 1 이상의 반응 채널 내에서 유동하는 유체의 유량 및 유속을 제어하기 위해 양압 또는 음압을 제공하도록 배치된 펌프를 더 포함할 수 있다. 또한, 상기 제1 히터 및 상기 제2 히터 사이에 광원이 배치되고, 상기 전극부에 전력을 공급하기 위한 전력 공급부, 상기 1 이상의 반응 채널 내에서 유동하는 유체의 유량 및 유속을 제어하기 위해 양압 또는 음압을 제공하도록 배치된 펌프, 및 상기 광원으로부터 방출되는 광을 검출하기 위한 광 검출부를 더 포함할 수 있다. 또한, 상기 전극부에 전력을 공급하기 위한 전력 공급부, 상기 1 이상의 반응 채널 내에서 유동하는 유체의 유량 및 유속을 제어하기 위해 양압 또는 음압을 제공하도록 배치된 펌프, 상기 PCR 칩에 광을 제공하도록 배치된 광 제공부, 및 상기 PCR 칩으로부터 방출되는 광을 수용하도록 배치된 광 검출부를 더 포함할 수 있다.In a sixth embodiment of the present invention, a heater group including one or more heaters, two or more heater groups, and the two or more heater groups include two or more heater units spaced apart from each other so that mutual heat exchange does not occur. A thermal block having a contact surface of a PCR chip containing at least one target sample thereon; An electrode unit having an electrode connected to supply electric power to heaters provided in the thermal block; And it provides a PCR device comprising a PCR chip according to a third embodiment of the present invention, arranged to be in contact with the heat block to enable heat exchange with one or more heaters provided in the heat block. In a fifth embodiment of the present invention, the thermal block may include two to four heater groups. In addition, the thermal block has two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature. In addition, the thermal block includes three heater groups, wherein the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group has the PCR extension step temperature. Or the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater The group may maintain the PCR extension step temperature, the second heater group may maintain the PCR denaturation step temperature, and the third heater group may maintain the PCR annealing step temperature. In addition, the thermal block may be implemented to have light transmittance. In addition, the heater provided in the heat block may include a light transmitting heating element. The apparatus may further include a power supply unit for supplying power to the electrode unit and a pump arranged to provide a positive pressure or a negative pressure to control a flow rate and a flow rate of the fluid flowing in the one or more reaction channels. In addition, a light source is disposed between the first heater and the second heater, a power supply for supplying power to the electrode portion, a positive pressure to control the flow rate and flow rate of the fluid flowing in the at least one reaction channel or The apparatus may further include a pump disposed to provide a negative pressure, and a light detector for detecting light emitted from the light source. In addition, a power supply for supplying power to the electrode portion, a pump arranged to provide a positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the one or more reaction channels, to provide light to the PCR chip The light providing unit may further include a light detecting unit disposed to receive the light emitted from the PCR chip.
본 발명의 제7 실시예는 구제역 감염이 의심되는 대상 시료를 본 발명의 제3 실시예에 따른 PCR 칩의 상기 1 이상의 반응 채널에 도입하여 PCR을 수행하는 단계; 및 상기 PCR 결과로부터 상기 대상 시료 중에 구제역 바이러스의 존재 유무를 확인하는 단계를 포함하는 구제역 검출 방법을 제공한다. 본 발명의 제6 실시예에 있어서, 상기 PCR 수행 단계는 본 발명의 제3 실시예에 따른 PCR 장치, 본 발명의 제4 실시예에 따른 PCR 장치, 및 본 발명의 제5 실시예에 따른 PCR 장치로 구성된 군으로부터 선택된 PCR 장치에서 수행될 수 있다.A seventh embodiment of the present invention includes the steps of performing a PCR by introducing a target sample suspected of foot and mouth infection into the one or more reaction channels of the PCR chip according to the third embodiment of the present invention; And it provides a foot-and-mouth disease detection method comprising the step of confirming the presence or absence of foot-and-mouth virus in the target sample from the PCR results. In a sixth embodiment of the present invention, the step of performing PCR is a PCR device according to a third embodiment of the present invention, a PCR device according to the fourth embodiment of the present invention, and a PCR according to the fifth embodiment of the present invention. And may be performed in a PCR device selected from the group consisting of devices.
본 발명의 일 실시예들에 따른 구제역의 혈청 타입별 원인 바이러스에 관한 유전자를 동시에 특이적으로 증폭할 수 있는 프라이머 세트, 이를 포함하는 PCR 칩, 이를 포함하는 PCR 장치, 및 이를 이용하는 구제역 검출 방법에 따르면, 구제역 발병 여부를 저렴한 비용으로 정확하고 신속하게 확인할 수 있어서 구제역 확산을 예방하고 이에 신속한 대응 조치를 취하는데에 크게 기여할 수 있다.In accordance with an embodiment of the present invention, a primer set capable of simultaneously specifically amplifying a gene related to a cause virus of each type of foot-and-mouth disease, a PCR chip comprising the same, a PCR device including the same, and a foot-and-mouth disease detecting method using the same According to the report, the outbreak of foot-and-mouth disease can be accurately and quickly confirmed at a low cost, thereby greatly contributing to preventing the spread of foot-and-mouth disease and taking prompt response thereto.
도 1은 본 발명의 제3 실시예에 따른 PCR 칩에 관한 도면이다.1 is a diagram of a PCR chip according to a third embodiment of the present invention.
도 2는 양면 접착제 또는 열 가소성 수지 또는 열 경화성 수지가 처리된 본 발명의 제3 실시예에 따른 PCR 칩에 관한 도면이다.2 is a diagram of a PCR chip according to a third embodiment of the present invention in which a double-sided adhesive, a thermoplastic resin, or a thermosetting resin is treated.
도 3a 내지 도 3c는 본 발명의 제4 실시예에 따른 PCR 장치에 관한 도면이다.3A to 3C are diagrams of a PCR device according to a fourth embodiment of the present invention.
도 4a 내지 도 4i는 본 발명의 제5 실시예에 따른 PCR 장치에 관한 도면이다.4A to 4I are diagrams illustrating a PCR device according to a fifth embodiment of the present invention.
도 5a 내지 도 5d는 본 발명의 제6 실시예에 따른 PCR 장치에 관한 도면이다.5A to 5D are diagrams of a PCR device according to a sixth embodiment of the present invention.
도 6 내지 11은 타사의 PCR 장치 및 본 발명의 제4 실시예에 따른 PCR 장치를 이용하여 혈청 타입별 구제역 바이러스 플라스미드 시료를 대상으로 하는 PCR 비교 결과를 나타낸다.Figures 6 to 11 show the results of PCR comparison of the foot-and-mouth disease virus plasmid samples for each serotype by using a third-party PCR device and a PCR device according to a fourth embodiment of the present invention.
이하, 첨부 도면을 참조하여 본 발명에 따른 실시예들을 상세하게 설명한다. 이하 설명은 본 발명에 따른 일 실시예들을 용이하게 이해하기 위한 것일 뿐이며, 보호범위를 제한하기 위한 것은 아니다.Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The following description is only for easily understanding the embodiments according to the present invention, but is not intended to limit the scope of protection.
본 발명의 제1 실시예는 서열번호 1의 염기 서열(CAC GCC GTG GGA CYA THC AGG A) 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머 및 서열번호 2의 염기 서열(GGG YTC RAA GAG RCG CCG GTA Y) 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머로 이루어진, 구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)로 구성된 군으로부터 1 이상 선택된 유전자를 검출하기 위한, 혈청 타입별 구제역(Foot and mouth disease, FMD) 검출용 프라이머 세트를 제공하고, 본 발명의 제2 실시예는 서열번호 3의 염기 서열(CAC GCC GTG GGA CCA TAC AGG A) 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머 및 서열번호 4의 염기 서열(GGG CTC AAA GAG ACG CCG GTA C) 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머로 이루어진, 구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)로 구성된 군으로부터 1 이상 선택된 유전자를 검출하기 위한, 혈청 타입별 구제역(Foot and mouth disease, FMD) 검출용 프라이머 세트를 제공한다.A first embodiment of the present invention is a primer comprising at least 15 consecutive nucleotides of the base sequence of SEQ ID NO: 1 (CAC GCC GTG GGA CYA THC AGG A) and the base sequence of SEQ ID NO: 2 (GGG YTC RAA GAG RCG CCG GTA Foot-and-mouth virus A (Genbank ID number: NC011450), foot-and-mouth virus O (Genbank ID number: NC004004), foot-and-mouth virus C (Genbank ID number: NC002554), foot-and-mouth disease, consisting of primers comprising at least 15 consecutive nucleotides of Y) By serotype, to detect one or more selected genes from the group consisting of virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451), and foot and mouth virus SAT 2 (Genbank ID number: NC011452) Provided is a primer set for detecting foot and mouth disease (FMD), the second embodiment of the present invention is a sequence of at least 15 consecutive of the base sequence of SEQ ID NO: 3 (CAC GCC GTG GGA CCA TAC AGG A) Foot-and-mouth virus A (Genbank ID number: NC011450), foot-and-mouth virus, consisting of a primer comprising a primer and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 4 (GGG CTC AAA GAG ACG CCG GTA C) O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 (Genbank ID number: NC011451), and foot and mouth virus SAT 2 (Genbank ID number: NC011452) provides a set of primers for detecting foot and mouth disease (FMD) for detecting one or more genes selected from the group consisting of.
상기 프라이머(primer)는 적합한 온도에서 적합한 완충액 내에서 적합한 조건(즉, 4종의 다른 뉴클레오시드 트리포스페이트 및 중합 반응 효소) 하에서 주형-지시 DNA 합성의 개시점으로 작용할 수 있는 단일 가닥의 올리고뉴클레오티드를 의미한다. 프라이머의 적합한 길이는 다양한 인자, 예를 들어, 온도와 프라이머의 용도에 따라 차이가 있지만 전형적으로 15 내지 30개의 뉴클레오티드이다. 짧은 프라이머는 주형과 충분히 안정된 혼성화 복합체를 형성하기 위하여 일반적으로 보다 낮은 온도를 요구할 수 있다. 이 경우, 전방향 프라이머(forward primer) 및 역방향 프라이머(reverse primer)는 중합 효소 연쇄 반응에 의해 증폭되는 주형의 일정한 부위의 3' 말단 및 5' 말단에 각각 결합하는 프라이머를 의미한다. 프라이머의 서열은 주형의 일부 서열과 완전하게 상보적인 서열을 가질 필요는 없으며, 주형과 혼성화되어 프라이머 고유의 작용을 할 수 있는 범위 내에서의 충분한 상보성을 가지면 족하다. 따라서, 본 발명의 제1 내지 제2 실시예에 따른 프라이머 세트는 주형인 뉴클레오티드 서열에 완전하게 상보적인 서열을 가질 필요는 없으며, 이 서열에 혼성화되어 프라이머 작용을 할 수 있는 범위 내에서 충분한 상보성을 가지면 족한 것이다. 이러한 프라이머의 설계는 주형이 되는 폴리뉴클레오티드의 염기 서열을 참조하여 당업자에 의해 용이하게 실시할 수 있으며, 예를 들어, 프라이머 설계용 프로그램(예를 들어, PRIMER 3, VectorNTI 프로그램)을 이용하여 할 수 있다. 한편, 본 발명의 일 실시예에 따른 프라이머는 주형의 한 부위에 혼성화 또는 어닐링되어, 이중쇄 구조를 형성한다. 이러한 이중쇄 구조를 형성하는 데 적합한 핵산 혼성화의 조건은 Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) 및 Haymes, B. D., 등, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985)에 개시되어 있다. 예를 들면, 상기 프라이머는 상기 서열번호 1 및 서열번호 2 중 어느 하나의 염기 서열 내의 15개 이상의 연속적인 뉴클레오티드를 포함할 수 있으며, 상기 프라이머는 상기 서열번호 1 및 서열번호 2 중 어느 하나의 염기 서열을 갖는 올리고뉴클레오티드일 수 있고, 상기 프라이머는 상기 서열번호 3 및 서열번호 4 중 어느 하나의 염기 서열 내의 15개 이상의 연속적인 뉴클레오티드를 포함할 수 있으며, 상기 프라이머는 상기 서열번호 3 및 서열번호 4 중 어느 하나의 염기 서열을 갖는 올리고뉴클레오티드일 수 있다. 한편, 본 발명의 제1 내지 제2 실시예에 따른 프라이머 세트의 PCR 산물의 크기는 90 내지 120 bp(base pair), 바람직하게는 92 bp(base pair), 어닐링 온도는 70 내지 72℃, 바람직하게는 서열번호 1 및 3의 프라이머는 72.7℃, 서열번호 2 및 4의 프라이머는 71.5℃, GC%는 50 내지 65%, 바람직하게는 서열번호 1 및 3의 프라이머는 63.6℃, 서열번호 2 및 4의 프라이머는 63.6℃로 설계되었다. 한편, 상기 서열번호 1 및 서열번호 2의 염기 서열 중, "Y"는 "t/u 또는 c"(피리미딘), "H"는 "a 또는 c 또는 t/u"(g 아님), "R"은 "g 또는 a"(푸린)를 의미한다.The primers are single-stranded oligonucleotides that can serve as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers at suitable temperatures. Means. Suitable lengths of the primers are typically 15 to 30 nucleotides, depending on various factors, such as temperature and the use of the primer. Short primers may generally require lower temperatures to form a hybridization complex that is sufficiently stable with the template. In this case, the forward primer and the reverse primer mean primers that bind to the 3 'end and the 5' end of a predetermined portion of the template to be amplified by the polymerase chain reaction. The sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range that can hybridize with the template to perform the primer-specific function. Therefore, the primer sets according to the first to second embodiments of the present invention do not need to have a sequence that is completely complementary to the nucleotide sequence that is a template, and sufficient complementarity within a range capable of hybridizing to this sequence to act as a primer. It is enough to have. The design of such primers can be easily carried out by those skilled in the art by referring to the nucleotide sequence of the polynucleotide to be a template, for example, using a primer design program (for example, PRIMER 3, VectorNTI program). have. On the other hand, the primer according to an embodiment of the present invention is hybridized or annealed to one site of the template to form a double-chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington, DC (1985). For example, the primer may comprise 15 or more consecutive nucleotides in the base sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer is the base of any one of SEQ ID NO: 1 and SEQ ID NO: 2 It may be an oligonucleotide having a sequence, wherein the primer may comprise at least 15 consecutive nucleotides in the base sequence of any one of SEQ ID NO: 3 and SEQ ID NO: 4, the primer is SEQ ID NO: 3 and SEQ ID NO: 4 It may be an oligonucleotide having any one of the base sequence. On the other hand, the size of the PCR product of the primer set according to the first to second embodiments of the present invention is 90 to 120 bp (base pair), preferably 92 bp (base pair), the annealing temperature is 70 to 72 ℃, preferably Preferably, primers of SEQ ID NOs: 1 and 3 are 72.7 ° C., primers of SEQ ID NOs: 2 and 4 are 71.5 ° C., GC% is 50-65%, preferably primers of SEQ ID NOs: 1 and 3 are 63.6 ° C., SEQ ID NOs: 2 and The primer of 4 was designed at 63.6 ° C. Meanwhile, in the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, "Y" is "t / u or c" (pyrimidine), "H" is "a or c or t / u" (not g), " R "means" g or a "(purine).
상기 프라이머 세트를 이용하여 구제역을 검출하는 방법은 다음과 같다. 먼저, 구제역 감염이 의심되는 대상 시료를 이하 상세하게 설명될 PCR 칩의 상기 1 이상의 반응 채널에 도입하여 PCR을 수행한다. 대상 시료라 함은 구제역에 감염되었을 것으로 예상되는 개체의 시료(샘플 또는 샘플 용액)를 의미하고, 예를 들어 배양된 세포, 혈액, 타액 등을 포함하나, 이에 제한되는 것은 아니다. 이 경우 구제역 감염이 의심되는 대상 시료는 상기 시료의 RNA로부터 cDNA를 합성하는 단계가 전제될 수 있다. 구제역 바이러스는 RNA를 게놈으로 하는 바, 이를 검출하기 위한 PCR을 수행하기 위해서 필요한 주형 DNA를 수득하기 위해, 바이러스의 게놈 RNA로부터 역전사를 수행하고, 이로부터 cDNA를 합성한다. 역전사 반응은 알려진 다양한 종류의 역전사 효소, 예를 들어 Invtrogen 사의 SuperScript 시리즈 및 이를 포함하는 키트를 통해 수행될 수 있다. 그 후, 상기 합성된 cDNA를 PCR 칩에 도입하여 실시간 PCR을 수행한다. 따라서, 상기 PCR 결과로부터 상기 대상 시료 중에 구제역 바이러스의 존재 유무를 확인할 수 있다. 이 경우 상기 PCR 수행 단계는 이하 상세하게 설명될 일련의 PCR 장치에서 수행될 수 있고, 더 나아가 실시간 PCR 장치를 이용할 경우 PCR 과정에서 증폭 산물에 표지된 형광 표지 인자를 감지하여 나타나는 곡선으로부터, PCR 증폭 산물이 일정량 증폭되었을 때의 사이클 수인 Ct 값(Ct value)을 계산함으로써 확인할 수 있다. 상기 Ct 값의 계산은 실시간 PCR 장치에 설치된 프로그램에 의해 자동으로 수행될 수 있음은 물론이다.The method of detecting foot-and-mouth disease using the primer set is as follows. First, PCR is performed by introducing a target sample suspected of foot and mouth infection into the one or more reaction channels of a PCR chip which will be described in detail below. Subject sample refers to a sample (sample or sample solution) of an individual expected to be infected with foot and mouth disease, and includes, but is not limited to, for example, cultured cells, blood, saliva, and the like. In this case, the target sample suspected of foot-and-mouth disease infection may be assumed to synthesize cDNA from the RNA of the sample. Foot-and-mouth disease viruses take RNA as a genome, perform reverse transcription from the virus's genomic RNA, and synthesize cDNA from it to obtain the template DNA necessary to perform PCR for detecting it. Reverse transcription reactions can be carried out through a variety of known reverse transcriptase enzymes, for example, the SuperScript series from Invtrogen, and kits comprising the same. Thereafter, the synthesized cDNA is introduced into a PCR chip to perform real time PCR. Therefore, the presence or absence of foot-and-mouth virus in the target sample can be confirmed from the PCR result. In this case, the step of performing PCR may be performed in a series of PCR devices to be described in detail below. Furthermore, when using a real-time PCR device, PCR amplification is performed from a curve that is detected by detecting a fluorescent labeling factor labeled on an amplification product during PCR. This can be confirmed by calculating the Ct value, which is the number of cycles when the product is amplified by a certain amount. The calculation of the Ct value may be automatically performed by a program installed in a real-time PCR apparatus.
도 1에 따르면, 본 발명의 제3 실시예에 따른 PCR 칩은 제1 판(11); 상기 제1 판(11) 상에 배치되고, 1 이상의 반응 채널(14)을 구비하는 제2 판(12); 및 상기 제2 판(12) 상에 배치되고, 상기 1 이상의 반응 채널(14)의 양 말단과 연결되되 개폐 가능하도록 구현된 유입부(15) 및 유출부(16)를 구비하는 제3 판(13)을 포함하는 것으로서, 상기 1 이상의 반응 채널(14) 내에 본 발명의 제1 내지 제2 실시예에 따른 프라이머 세트를 포함한다.1, a PCR chip according to a third embodiment of the present invention comprises: a first plate 11; A second plate (12) disposed on the first plate (11) and having one or more reaction channels (14); And a third plate disposed on the second plate 12 and having an inlet portion 15 and an outlet portion 16 connected to both ends of the one or more reaction channels 14 to be opened and closed. 13), comprising in said at least one reaction channel 14 a primer set according to the first to second embodiments of the invention.
상기 PCR 칩(10)의 반응 채널(14) 내에는 상기 본 발명의 제1 내지 제2 실시예에 따른 프라이머 세트 이외에 삼인산화데옥시리보뉴클레오티드(deoxyribonucleotide triphosphates, dNTP), 구체적으로 dATP, dCTP, dGTP, 및 dTTP를 포함하는 혼합물, DNA 중합효소, 검출 가능한 표지, 및 PCR 완충액(PCR buffer)를 더 포함할 수 있다. 상기 DNA 중합 효소는 예를 들어, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis 또는 Pyrococcus furiosus (Pfu)로부터 수득한 열 안정성 DNA 중합 효소일 수 있다. 상기 검출 가능한 표지는 Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 및 티아졸 오렌지(thiazole orange)로 이루어진 군으로부터 선택될 수 있다. 상기 PCR 완충액은 증폭 반응의 pH를 조절함으로써 증폭 반응의 하나 이상의 구성 요소의 안정성, 활성, 및/또는 수명을 변형시키는 증폭 반응에 첨가되는 화합물로서, 이러한 완충액들은 알려져 있으며, 예를 들어, Tris, Tricine, MOPS, 또는 HEPES일 수 있으나, 이에 제한되는 것은 아니다. 상기 PCR 칩(10)은 상기 샘플 용액을 도입하기 위한 유입부(15), 핵산 증폭 반응을 완료한 샘플 용액을 배출하기 위한 유출부(16) 및 증폭하고자 하는 핵산을 포함하는 샘플 용액이 수용된 1 이상의 반응 채널(14)를 포함할 수 있다. 상기 PCR 칩(10)이 PCR 장치의 열 블록에 접촉하는 경우 상기 열 블록에서 발생하는 열은 상기 PCR 칩(10)에 전달되고, 상기 PCR 칩(10)의 반응 채널(14)에 포함된 샘플 용액은 가열되거나 냉각되어 일정 온도가 유지될 수 있다. 또한, 상기 PCR 칩(10)은 전체적으로 평면 형상을 가질 수 있으나, 이에 제한되는 것은 아니다. 또한, 상기 PCR 칩(10)은 PCR 장치의 칩 홀더에 장착된 상태로 상기 열 블록에 접촉 배치될 수도 있다. 따라서, 상기 PCR 칩(10)이 상기 열 블록의 일 면에 배치된다는 것은 상기 PCR 칩(10)이 상기 칩 홀더에 장착된 상태로 상기 열 블록에 접촉 배치되는 것을 포함한다. 또한, 상기 PCR 칩(10)은 플라스틱 재질로 구현되되, 광 투과성을 갖도록 구현될 수 있다. 상기 PCR 칩(10)은 플라스틱 재질을 사용하여, 플라스틱 두께 조절만으로 열 전달 효율을 증대시킬 수 있고, 제작 공정이 단순하여 제조 비용을 절감할 수 있다. 또한 상기 PCR 칩(10)은 전체적으로 광 투과성을 구비할 수 있기 때문에 상기 열 블록의 일 면에 배치된 상태에서 직접적으로 광 조사가 가능하여 실시간으로 핵산 증폭 여부 및 증폭 정도를 측정 및 분석할 수 있다.In the reaction channel 14 of the PCR chip 10, deoxyribonucleotide triphosphates (dNTP), specifically dATP, dCTP, dGTP, in addition to the primer sets according to the first to second embodiments of the present invention, And, a mixture comprising dTTP, a DNA polymerase, a detectable label, and a PCR buffer. The DNA polymerase may be, for example, a heat stable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus furiosus (Pfu). The detectable label is Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2 , Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43 , SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 and thiazole orange. The PCR buffer is a compound that is added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction. Such buffers are known, for example Tris, Tricine, MOPS, or HEPES, but is not limited thereto. The PCR chip 10 includes an inlet 15 for introducing the sample solution, an outlet 16 for discharging the sample solution having completed the nucleic acid amplification reaction, and a sample solution containing the nucleic acid to be amplified. The reaction channel 14 may be included. When the PCR chip 10 contacts the thermal block of the PCR device, heat generated in the thermal block is transferred to the PCR chip 10, and a sample included in the reaction channel 14 of the PCR chip 10. The solution may be heated or cooled to maintain a constant temperature. In addition, the PCR chip 10 may have a flat shape as a whole, but is not limited thereto. In addition, the PCR chip 10 may be disposed in contact with the thermal block while being mounted on the chip holder of the PCR device. Thus, the arrangement of the PCR chip 10 on one surface of the column block includes contact arrangement of the PCR chip 10 with the column block while being mounted on the chip holder. In addition, the PCR chip 10 is implemented with a plastic material, it may be implemented to have a light transmission. The PCR chip 10 may use a plastic material to increase the heat transfer efficiency only by adjusting the thickness of the plastic, and the manufacturing process may be simplified to reduce the manufacturing cost. In addition, since the PCR chip 10 may be provided with light transmittance as a whole, light can be directly irradiated in a state in which it is disposed on one surface of the heat block, thereby measuring and analyzing nucleic acid amplification and amplification degree in real time. .
상기 제1 판(11)은 상기 제2 판(12) 하부 면에 배치된다. 상기 제1 판(11)이 상기 제2 판(920)의 하부 면에 접착 배치됨으로써 상기 1 이상의 반응 채널(14)은 일종의 PCR 반응 챔버를 형성한다. 또한, 상기 제1 판(11)은 다양한 재질로 구현될 수 있으나, 바람직하게는 폴리디메틸실옥산(polydimethylsiloxane, PDMS), 사이클로올레핀코폴리머(cycle olefin copolymer, COC), 폴리메틸메타크릴레이트(polymethylmetharcylate, PMMA), 폴리카보네이트(polycarbonate, PC), 폴리프로필렌카보네이트(polypropylene carbonate, PPC), 폴리에테르설폰(polyether sulfone, PES), 및 폴리에틸렌텔레프탈레이트(polyethylene terephthalate, PET), 및 그의 조합물로 구성된 군으로부터 선택되는 재질일 수 있다. 또한, 상기 제1 판(11)의 상단 면은 친수성 물질(17)이 처리되어 PCR을 원활하게 수행할 수 있다. 상기 친수성 물질(17)의 처리에 의해 상기 제1 판(11) 상에 친수성 물질(17)을 포함하는 단일 층이 형성될 수 있다. 상기 친수성 물질은 다양한 물질일 수 있으나, 바람직하게는 카르복시기(-COOH), 아민기(-NH2), 히드록시기(-OH), 및 술폰기(-SH)로 구성된 군으로부터 선택되는 것일 수 있고, 상기 친수성 물질의 처리는 당 업계에 공지된 방법에 따라 수행할 수 있다.The first plate 11 is disposed on the bottom surface of the second plate 12. The first plate 11 is bonded to the lower surface of the second plate 920 so that the one or more reaction channels 14 form a kind of PCR reaction chamber. In addition, the first plate 11 may be made of various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from. In addition, the hydrophilic material 17 is treated on the upper surface of the first plate 11 to perform PCR smoothly. By treatment of the hydrophilic material 17, a single layer comprising hydrophilic material 17 may be formed on the first plate 11. The hydrophilic material may be a variety of materials, but preferably may be selected from the group consisting of carboxyl group (-COOH), amine group (-NH2), hydroxy group (-OH), and sulfone group (-SH), Treatment of the hydrophilic material can be carried out according to methods known in the art.
상기 제2 판(12)은 상기 제1 판(11) 상부 면에 배치된다. 상기 제2 판(920)은 1 이상의 반응 채널(14)을 포함한다. 상기 반응 채널(14)은 상기 제3 판(13)에 형성된 유입부(15)과 유출부(16)에 대응되는 부분과 연결되어 일종의 PCR 반응 챔버를 형성한다. 따라서, 상기 반응 채널(921)에 증폭하고자 하는 샘플 용액이 도입된 후 PCR이 진행된다. 또한, 상기 반응 채널(14)은 PCR 장치의 사용 목적 및 범위에 따라 2 이상 존재할 수 있고, 도 1에 따르면, 6개의 반응 채널(14)이 예시되고 있다. 또한, 상기 제2 판(12)은 다양한 재질로 구현될 수 있으나, 바람직하게는 폴리메틸메타크릴레이트(polymethylmethacrylate, PMMA), 폴리카보네이트(polycarbonate, PC), 사이클로올레핀 코폴리머(cycloolefin copolymer, COC), 폴리아미드(polyamide, PA), 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리페닐렌 에테르(polyphenylene ether, PPE), 폴리스티렌(polystyrene, PS), 폴리옥시메틸렌(polyoxymethylene, POM), 폴리에테르에테르케톤(polyetheretherketone, PEEK), 폴리테트라프로오르에틸렌(polytetrafluoroethylene, PTFE), 폴리비닐클로라이드(polyvinylchloride, PVC), 폴리비닐리덴 플로라이드(polyvinylidene fluoride, PVDF), 폴리부틸렌테레프탈레이트(polybutyleneterephthalate, PBT), 불소화에틸렌프로필렌(fluorinated ethylenepropylene, FEP), 퍼플로로알콕시알칸(perfluoralkoxyalkane, PFA), 및 그의 조합물로 구성된 군으로부터 선택되는 열 가소성 수지 또는 열 경화성 수지 재질일 수 있다. 또한, 상기 제2 판(12)의 두께는 다양할 수 있으나, 100 ㎛ 내지 200 ㎛에서 선택될 수 있다. 또한, 상기 반응 채널(14)의 폭과 길이는 다양할 수 있으나, 바람직하게는 상기 반응 채널(14)의 폭은 0.5 mm 내지 3 mm에서 선택되고, 상기 관통 개구 채널(14)의 길이는 20 mm 내지 40 mm에서 선택될 수 있다. 또한, 상기 제2 판(12) 내벽은 DNA, 단백질(protein) 흡착을 방지하기 위해 실란(silane) 계열, 보바인 시럼 알부민(Bovine Serum Albumin, BSA) 등의 물질로 코팅할 수 있고, 상기 물질의 처리는 당 업계에 공지된 방법에 따라 수행될 수 있다.The second plate 12 is disposed on an upper surface of the first plate 11. The second plate 920 includes one or more reaction channels 14. The reaction channel 14 is connected to portions corresponding to the inlet 15 and the outlet 16 formed in the third plate 13 to form a kind of PCR reaction chamber. Therefore, PCR is performed after the sample solution to be amplified is introduced into the reaction channel 921. In addition, the reaction channel 14 may be present in two or more according to the purpose and scope of use of the PCR apparatus, and according to FIG. 1, six reaction channels 14 are illustrated. In addition, the second plate 12 may be made of various materials, but preferably, polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (cycloolefin copolymer, COC) , Polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (polybutylene terephthalate) , PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), and combinations thereof It is chosen or a thermoplastic resin may be a thermosetting resin material. In addition, the thickness of the second plate 12 may vary, but may be selected from 100 μm to 200 μm. In addition, the width and length of the reaction channel 14 may vary, but preferably the width of the reaction channel 14 is selected from 0.5 mm to 3 mm, the length of the through-opening channel 14 is 20 can be selected from mm to 40 mm. In addition, the inner wall of the second plate 12 may be coated with a material such as silane-based and Bovine Serum Albumin (BSA) to prevent DNA and protein adsorption. The treatment of can be carried out according to methods known in the art.
상기 제3 판(13)은 상기 제2 판(12) 상에 배치된다. 상기 제3 판(13)은 상기 제2 판(12)에 형성된 1 이상의 반응 채널(921) 상의 일 영역에 형성된 유입부(15) 및 다른 일 영역에 형성된 유출부(16)를 구비한다. 상기 유입부(15)는 증폭하고자 하는 핵산을 포함하는 샘플 용액 등이 유입되는 부분이다. 상기 유출부(16)는 PCR이 종료된 후 샘플 용액 등이 유출되는 부분이다. 따라서, 상기 제3 판(13)은 이하 언급할 제2 판(12)에 형성된 1 이상의 반응 채널(14)을 커버하되, 상기 유입부(15) 및 유출부(16)는 상기 반응 채널(14)의 유입부 및 유출부 역할을 수행하게 된다. 또한, 상기 제3 판(13)은 다양한 재질로 구현될 수 있지만, 바람직하게는 폴리디메틸실옥산(polydimethylsiloxane, PDMS), 사이클로올레핀코폴리머(cycle olefin copolymer, COC), 폴리메틸메타크릴레이트(polymethylmetharcylate, PMMA), 폴리카보네이트(polycarbonate, PC), 폴리프로필렌카보네이트(polypropylene carbonate, PPC), 폴리에테르설폰(polyether sulfone, PES), 및 폴리에틸렌텔레프탈레이트(polyethylene terephthalate, PET), 및 그의 조합물로 구성된 군으로부터 선택되는 재질일 수 있다. 또한, 상기 유입부(15)은 다양한 크기를 구비할 수 있으나, 바람직하게는 지름 1.0 mm 내지 3.0 mm에서 선택될 수 있다. 또한, 상기 유출부(16)는 다양한 크기를 구비할 수 있으나, 바람직하게는 지름 1.0 mm 내지 1.5 mm에서 선택될 수 있다. 또한, 상기 유입부(15) 및 유출부(16)는 별도의 커버 수단(도시되지 않음)을 구비하여, 상기 반응 채널(14) 내에서 샘플 용액에 대한 PCR이 진행될 때 샘플 용액이 누출되는 것을 방지할 수 있다. 상기 커버 수단은 다양한 형상, 크기 또는 재질로서 구현될 수 있다. 또한, 상기 제3 판의 두께는 다양할 수 있으나, 바람직하게는 0.1 mm 내지 2.0 mm에서 선택될 수 있다. 또한, 상기 유입부(15) 및 유출부(16)는 2 이상 존재할 수 있다.The third plate 13 is disposed on the second plate 12. The third plate 13 has an inlet 15 formed in one region on one or more reaction channels 921 formed in the second plate 12 and an outlet 16 formed in the other region. The inlet 15 is a portion into which a sample solution containing a nucleic acid to be amplified is introduced. The outlet 16 is a portion where the sample solution and the like flows out after the PCR is completed. Thus, the third plate 13 covers one or more reaction channels 14 formed in the second plate 12, which will be discussed below, wherein the inlet 15 and outlet 16 are the reaction channels 14 ) Will serve as the inlet and outlet. In addition, the third plate 13 may be made of various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from. In addition, the inlet 15 may have various sizes, but preferably may be selected from 1.0 mm to 3.0 mm in diameter. In addition, the outlet 16 may have a variety of sizes, but preferably may be selected from 1.0 mm to 1.5 mm in diameter. In addition, the inlet part 15 and the outlet part 16 are provided with separate cover means (not shown), so that the sample solution leaks when PCR is performed on the sample solution in the reaction channel 14. It can prevent. The cover means may be implemented in various shapes, sizes or materials. In addition, the thickness of the third plate may vary, but may preferably be selected from 0.1 mm to 2.0 mm. In addition, the inlet 15 and the outlet 16 may be present at least two.
도 2에 따르면, 상기 PCR 칩(10)은 기계적 가공을 통해 유입부(15) 및 유출부(16)를 형성하여 제3 판(13)을 제공하는 단계; 상기 제3 판(13)의 하부면과 대응되는 크기를 갖는 판재에 상기 제3 판(13)의 유입부(15)와 대응되는 부분으로부터 상기 제3 판(13)의 유출부(16)에 대응되는 부분까지 기계적 가공을 통해 1 이상의 반응 채널(14)을 형성하여 제2 판(12)을 제공하는 단계; 상기 제2 판(12)의 하부면과 대응되는 크기를 갖는 판재의 상부면에 표면 처리 가공을 통해 친수성 물질(17)로 구현된 표면을 형성하여 제1 판(11)을 제공하는 단계; 및 상기 제3 판(13)의 하부면을 상기 제2 판(12)의 상부면에 접합 공정을 통해 접합하고, 상기 제2 판(12)의 하부면을 상기 제1 판(11)의 상부면에 접합 공정을 통해 접합하는 단계를 포함하는 방법에 의해 용이하게 제조될 수 있다. 상기 제3 판(13)의 유입부(15) 및 유출부(16), 및 상기 제2 판(12)의 반응 채널(14)은 사출성형, 핫-엠보싱(hot-embossing), 캐스팅(casting), 및 레이저 어블레이션(laser ablation)으로 구성된 군으로부터 선택되는 가공 방법에 의해 형성될 수 있다. 또한, 상기 제1 판(11) 표면의 친수성 물질(17)은 산소 및 아르곤 플라즈마 처리, 코로나 방전 처리, 및 계면 활성제 도포로 구성된 군으로부터 선택되는 방법에 의해 처리될 수 있고 당 업계에 공지된 방법에 따라 수행될 수 있다. 또한, 상기 제3 판(13)의 하부 면과 상기 제2 판(12)의 상부면, 및 상기 제2 판(12)의 하부 면과 상기 제1 판(11)의 상부면은 열 접합, 초음파 융착, 용매 접합 공정에 의해 접착될 수 있고 당 업계에 공지된 방법에 따라 수행될 수 있다. 상기 제3 판(13)과 제2 판(12) 사이 및 상기 제2 판(12)과 제3 판(13) 사이에는 양면 접착제 또는 열가소성 수지 또는 열 경화성 수지(18)가 처리될 수 있다.According to FIG. 2, the PCR chip 10 forms a inlet 15 and an outlet 16 through mechanical processing to provide a third plate 13; From the portion corresponding to the inlet portion 15 of the third plate 13 to the plate portion having a size corresponding to the lower surface of the third plate 13 to the outlet portion 16 of the third plate 13 Forming one or more reaction channels 14 by mechanical machining to the corresponding portion to provide a second plate 12; Providing a first plate (11) by forming a surface made of a hydrophilic material (17) through surface treatment on an upper surface of a plate having a size corresponding to a lower surface of the second plate (12); And bonding the lower surface of the third plate 13 to the upper surface of the second plate 12 through a bonding process, and the lower surface of the second plate 12 to the upper portion of the first plate 11. It can be easily produced by a method comprising the step of bonding to the surface through a bonding process. The inlet 15 and outlet 16 of the third plate 13 and the reaction channel 14 of the second plate 12 are injection molded, hot-embossing and casting. ), And a processing method selected from the group consisting of laser ablation. In addition, the hydrophilic material 17 on the surface of the first plate 11 may be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application and are known in the art. Can be performed according to. In addition, the lower surface of the third plate 13 and the upper surface of the second plate 12, and the lower surface of the second plate 12 and the upper surface of the first plate 11 are thermally bonded, It can be adhered by ultrasonic fusion, solvent bonding processes and can be carried out according to methods known in the art. A double-sided adhesive or a thermoplastic or thermosetting resin 18 may be treated between the third plate 13 and the second plate 12 and between the second plate 12 and the third plate 13.
이하, 본 발명의 제4 실시예에 따른 PCR 칩이 구동되는 PCR 장치에 관하여 설명한다. PCR 장치라 함은 특정 염기 서열을 갖는 핵산을 증폭하는 PCR(Polymerase Chain Reaction)에 사용하기 위한 장치이다. 예를 들어, 특정 염기 서열을 갖는 DNA(deoxyribonucleic acid)를 증폭하기 위한 PCR 장치는 이중 가닥의 DNA를 포함하는 샘플 용액을 특정 온도, 예를 들어 약 95℃로 가열하여 상기 이중 가닥의 DNA를 단일 가닥의 DNA로 분리하는 변성 단계(denaturing step), 상기 샘플 용액에 증폭하고자 하는 특정 염기 서열과 상보적인 서열을 갖는 올리고뉴클레오티드(oligonucleotide) 프라이머를 제공하고, 상기 분리된 단일 가닥의 DNA와 함께 특정 온도, 예를 들어 55℃로 냉각하여 상기 단일 가닥의 DNA의 특정 염기 서열에 상기 프라이머를 결합시켜 부분적인 DNA-프라이머 복합체를 형성하는 어닐링 단계(annealing step), 및 상기 어닐링 단계 이후 상기 샘플 용액을 적정 온도, 예를 들어 72℃로 유지하여 DNA 중합효소(polymerase)에 의해 상기 부분적인 DNA-프라이머 복합체의 프라이머를 기초로 이중 가닥의 DNA를 형성하는 연장 (혹은 증폭) 단계(extension step)를 수행하고, 상기 3 단계를 예를 들어, 20회 내지 40회로 반복함으로써 상기 특정 염기 서열을 갖는 DNA를 기하급수적으로 증폭할 수 있다. 또한, 경우에 따라, PCR 장치는 상기 어닐링 단계와 상기 연장 (혹은 증폭) 단계를 동시에 수행할 수 있고, 이 경우 PCR 장치는 상기 연장 단계와 상기 어닐링 및 연장 (혹은 증폭) 단계로 구성된 2 단계를 수행함으로써, 제1 순환을 완성할 수도 있다. 따라서, 본 명세서에 있어서, PCR 장치는 상기 단계들을 수행하기 위한 모듈들을 포함하는 장치를 말하며, 본 명세서에 기재되지 아니한 세부적인 모듈들은 PCR을 수행하기 위한 종래 기술 중 개시되고 자명한 범위에서 모두 구비하고 있는 것을 전제로 한다.Hereinafter, a PCR device for driving a PCR chip according to a fourth embodiment of the present invention will be described. The PCR device is a device for use in PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific base sequence. For example, a PCR device for amplifying deoxyribonucleic acid (DNA) having a specific nucleotide sequence may be used to heat a sample solution containing double stranded DNA to a specific temperature, for example about 95 ° C. A denaturing step of separating DNA into strands, providing an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified in the sample solution, and a specific temperature with the separated single strand of DNA. For example, an annealing step in which the primer is coupled to a specific base sequence of the single strand of DNA by cooling to 55 ° C. to form a partial DNA-primer complex, and the sample solution is titrated after the annealing step. Temperature based on primers of the partial DNA-primer complex by DNA polymerase, for example at 72 ° C. By performing an extension (or amplification) step of forming a double strand of DNA and repeating the three steps, for example, 20 to 40 times, the DNA having the specific base sequence can be exponentially amplified. have. Also, in some cases, the PCR device may simultaneously perform the annealing step and the extension (or amplification) step, and in this case, the PCR device may perform two steps including the extension step and the annealing and extension (or amplification) step. By performing, the first circulation may be completed. Therefore, in the present specification, a PCR device refers to a device including modules for performing the above steps, and detailed modules not described herein are all provided in the prior art and obvious range for performing PCR. It is assumed that you are doing.
도 3a 내지 도 3c에 따르면, 본 발명의 제4 실시예에 따른 PCR 장치가 상세하게 설명된다.3A to 3C, a PCR device according to a fourth embodiment of the present invention will be described in detail.
도 3a에 따르면, 본 발명의 제4 실시예에 따른 PCR 장치는 기판(400a) 상에 배치된 제1 열 블록(100a); 상기 기판(400a) 상에 상기 제1 열 블록(100a)과 이격 배치된 제2 열 블록(200a); 및 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 위로 구동 수단(500a)에 의해 좌우 및/또는 상하 이동 가능하고, 상기 본 발명의 제3 실시예에 따른 PCR 칩(10)이 장착된 칩 홀더(300a)를 포함한다.According to FIG. 3A, a PCR device according to a fourth embodiment of the present invention includes a first row block 100a disposed on a substrate 400a; A second thermal block 200a spaced apart from the first thermal block 100a on the substrate 400a; And move left, right, and / or up and down by the driving means 500a over the first row block 100a and the second row block 200a, and the PCR chip 10 according to the third embodiment of the present invention is And a mounted chip holder 300a.
상기 기판(400a)은 상기 제1 열 블록(100a) 및 제2 열 블록(200a)의 가열 및 온도 유지로 인해 그 물리적 및/또는 화학적 성질이 변하지 않고, 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 사이에서 상호 열 교환이 일어나지 않도록 하는 재질을 갖는 모든 물질을 포함한다. 예를 들어, 상기 기판(400a)은 플라스틱 등의 재질을 포함하거나 그러한 재질로 구성될 수 있다.The substrate 400a does not change its physical and / or chemical properties due to heating and temperature maintenance of the first thermal block 100a and the second thermal block 200a, and the first thermal block 100a and the first thermal block 100a and the second thermal block 200a do not change. It includes all materials having a material such that mutual heat exchange does not occur between the two heat blocks 200a. For example, the substrate 400a may include or be made of a material such as plastic.
상기 제1 열 블록(100a) 및 제2 열 블록(200a)은 핵산을 증폭하기 위한 변성 단계, 어닐링 단계 및 연장 (혹은 증폭) 단계를 수행하기 위한 온도를 유지하기 위한 것이다. 따라서 상기 제1 열 블록(100a) 및 제2 열 블록(200a)은 상기 각 단계들에 요구되는 필요한 온도를 제공하고, 이를 유지하기 위한 다양한 모듈을 포함하거나 또는 그러한 모듈과 구동가능하게 연결될 수 있다. 따라서, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)가 상기 각 열 블록(100a, 200a)의 일 면에 접촉되는 경우 상기 제1 열 블록(100a) 및 제2 열 블록(200a)은 상기 PCR 칩(10)과의 접촉면을 전체적으로 가열 및 온도 유지할 수 있어서, 상기 PCR 칩(10) 내의 샘플 용액을 균일하게 가열 및 온도 유지할 수 있다. 종래 단일 열 블록을 사용하는 PCR 장치는 상기 단일 열 블록에서의 온도 변화율이 초당 3 내지 7℃ 범위 내에서 이루어지는데 반해, 본 발명의 제3 실시예에 따른 2개의 열 블록을 포함하는 PCR 장치는 각각의 열 블록(100a, 200a)에서의 온도 변화율이 초당 20 내지 40℃ 범위 내에서 이루어져 PCR 진행 시간을 크게 단축시킬 수 있다.The first row block 100a and the second row block 200a are for maintaining a temperature for performing a denaturation step, annealing step and extension (or amplification) step for amplifying the nucleic acid. Thus, the first thermal block 100a and the second thermal block 200a may include or be operably connected with various modules for providing and maintaining the required temperature required for the respective steps. . Therefore, when the chip holder 300a on which the PCR chip 10 is mounted is in contact with one surface of each of the row blocks 100a and 200a, the first row block 100a and the second row block 200a are Since the contact surface with the PCR chip 10 can be heated and maintained at a temperature as a whole, the sample solution in the PCR chip 10 can be heated and maintained at a uniform temperature. In a conventional PCR apparatus using a single heat block, the temperature change rate in the single heat block is within a range of 3 to 7 ° C per second, whereas a PCR device including two heat blocks according to a third embodiment of the present invention The rate of temperature change in each of the thermal blocks 100a and 200a may be within a range of 20 to 40 ° C. per second to greatly shorten the PCR progress time.
상기 제1 열 블록(100a) 및 제2 열 블록(200a)은 그 내부에 열선(도시되지 않음)이 배치될 수 있다. 상기 열선은 상기 변성 단계, 어닐링 단계 및 연장 (혹은 증폭) 단계를 수행하기 위한 온도를 유지하도록 다양한 열원과 구동가능하게 연결될 수 있고, 상기 열선의 온도를 모니터링하기 위한 다양한 온도 센서와 구동가능하게 연결될 수 있다. 상기 열선은 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 내부 온도를 전체적으로 일정하게 유지하기 위해 각각의 열 블록(100a, 200a) 면의 중심점을 기준으로 상하 및/또는 좌우 방향으로 대칭되도록 배치될 수 있다. 상기 상하 및/또는 좌우 방향으로 대칭된 열선의 배치는 다양할 수 있다. 또한, 상기 제1 열 블록(100a) 및 제2 열 블록(200a)은 그 내부에 박막 히터(thin film heater, 도시되지 않음)가 배치될 수도 있다. 상기 박막 히터는 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 내부 온도를 전체적으로 일정하게 유지하기 위해 각각의 열 블록(100a, 200a) 면의 중심점을 기준으로 상하 및/또는 좌우 방향으로 일정한 간격으로 이격 배치될 수 있다. 상기 상하 및/또는 좌우 방향으로 일정한 박막 히터의 배치는 다양할 수 있다.Hot wires (not shown) may be disposed in the first row block 100a and the second row block 200a. The heating wire may be operably connected with various heat sources to maintain a temperature for performing the denaturing, annealing and extending (or amplifying) steps, and may be operably connected with various temperature sensors for monitoring the temperature of the heating wire. Can be. The heating wires are vertically and / or horizontally based on the center point of the surface of each of the heat blocks 100a and 200a in order to maintain a constant internal temperature of the first and second heat blocks 100a and 200a. It may be arranged to be symmetrical. The arrangement of the hot wires symmetrical in the vertical direction and / or the horizontal direction may vary. In addition, a thin film heater (not shown) may be disposed in the first thermal block 100a and the second thermal block 200a. The thin-film heater is vertically and / or horizontally based on a center point of each of the heat block 100a and 200a in order to maintain a constant internal temperature of the first and second heat blocks 100a and 200a. May be spaced apart at regular intervals. The arrangement of the thin film heater that is constant in the vertical and / or horizontal directions may vary.
상기 제1 열 블록(100a) 및 제2 열 블록(200a)은 동일한 면적에 대한 고른 열 분포 및 신속한 열 전달을 위해 금속 재질, 예를 들어 알루미늄 재질을 포함하거나 또는 알루미늄 재질로 구성될 수 있다.The first heat block 100a and the second heat block 200a may include a metal material, for example, aluminum or may be made of aluminum for even heat distribution and rapid heat transfer over the same area.
상기 제1 열 블록(100a)은 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행하기 위한 적정 온도를 유지하도록 구현될 수 있다. 예를 들어, 본 발명의 제3 실시예에 따른 PCR 장치의 제1 열 블록(100a)은 50℃ 내지 100℃를 유지할 수 있고, 바람직하게는 상기 제1 열 블록(100a)에서 상기 변성 단계를 수행하는 경우 90℃ 내지 100℃를 유지할 수 있고, 바람직하게는 95℃를 유지할 수 있으며, 상기 제1 열 블록(100a)에서 상기 어닐링 및 연장 (혹은 증폭) 단계를 수행하는 경우에는 55℃ 내지 75℃를 유지할 수 있고, 바람직하게는 72℃를 유지할 수 있다. 다만, 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행할 수 있는 온도라면 이에 제한되는 것은 아니다. 상기 제2 열 블록(200a)은 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행하기 위한 적정 온도를 유지하도록 구현될 수 있다. 예를 들어, 본 발명의 제3 실시예에 따른 PCR 장치의 제2 열 블록(200a)은 상기 제2 열 블록(200a)에서 상기 변성 단계를 수행하는 경우 90℃ 내지 100℃를 유지할 수 있고, 바람직하게는 95℃를 유지할 수 있으며, 상기 제2 열 블록에서 상기 어닐링 및 연장 (혹은 증폭) 단계를 수행하는 경우에는 55℃ 내지 75℃를 유지할 수 있고, 바람직하게는 72℃를 유지할 수 있다. 다만, 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행할 수 있는 온도라면 이에 제한되는 것은 아니다. 따라서, 본 발명의 제3 실시예에 따르면, 상기 제1 열 블록(100a)은 PCR의 변성 단계 온도 (denaturing temperature)를 유지할 수 있으며, 변성 단계 온도가 90℃보다 낮으면 PCR의 주형이 되는 핵산의 변성이 일어나 효율이 떨어져 PCR 효율이 떨어지거나 반응이 일어나지 않을 수 있고, 변성 단계 온도가 100℃보다 높아지면 PCR에 이용되는 효소가 활성을 잃게 되므로, 상기 변성 단계 온도는 90℃ 내지 100℃일 수 있고, 바람직하게는 95℃일 수 있다. 또한, 본 발명의 제3 실시예에 따르면, 상기 제2 열 블록(200a)은 PCR의 어닐링 및 연장 (혹은 증폭) 단계 온도(annealing/extension temperature)를 유지할 수 있다. 연장 (혹은 증폭) 단계 온도가 55℃보다 낮으면 PCR 산물의 특이성(specificity)이 떨어질 수 있고, 어닐링 및 연장 (혹은 증폭) 단계 온도가 74℃보다 높으면 프라이머에 의한 연장이 일어나지 않을 수 있기 때문에 PCR 효율이 떨어지게 되므로 상기 어니링 및 연장 (혹은 증폭) 단계 온도는 55℃ 내지 75℃일 수 있고, 바람직하게는 72℃일 수 있다.The first thermal block 100a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps. For example, the first row block 100a of the PCR apparatus according to the third embodiment of the present invention may maintain 50 ° C. to 100 ° C., and preferably, the denaturation step is performed in the first row block 100 a. If performed, the temperature may be maintained at 90 ° C. to 100 ° C., preferably at 95 ° C., and may be 55 ° C. to 75 ° when the annealing and extension (or amplification) steps are performed in the first thermal block 100 a. ℃ can be maintained, preferably 72 ℃. However, the temperature of the denaturation step or the annealing and extension (or amplification) step is not limited thereto. The second row block 200a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps. For example, the second row block 200a of the PCR apparatus according to the third embodiment of the present invention may maintain 90 ° C. to 100 ° C. when the denaturation step is performed in the second row block 200 a. Preferably, the temperature may be maintained at 95 ° C., and may be maintained at 55 ° C. to 75 ° C., preferably at 72 ° C., when the annealing and extension (or amplification) steps are performed in the second heat block. However, the temperature of the denaturation step or the annealing and extension (or amplification) step is not limited thereto. Therefore, according to the third embodiment of the present invention, the first row block 100a may maintain the denaturing temperature of the PCR, and if the denaturation temperature is lower than 90 ° C, the nucleic acid is a template of the PCR. When the denaturation occurs, the efficiency is low, the PCR efficiency may not be reduced, or the reaction may not occur. When the denaturation step temperature is higher than 100 ° C., the enzyme used for PCR loses activity, and thus, the denaturation step temperature is 90 ° C. to 100 ° C. And preferably 95 ° C. In addition, according to the third embodiment of the present invention, the second row block 200a may maintain annealing / extension temperature of annealing and extension (or amplification) of PCR. If the extension (or amplification) step temperature is lower than 55 ° C., the specificity of the PCR product may be degraded. If the annealing and extension (or amplification) step temperature is higher than 74 ° C., the PCR may not occur. Since the efficiency is lowered, the annealing and extension (or amplification) step temperature may be 55 ° C to 75 ° C, preferably 72 ° C.
상기 제1 열 블록(100a)과 제2 열 블록(200a)은 상호 열 교환이 일어나지 않도록 미리 결정된 거리로 이격 배치될 수 있다. 이에 따라, 상기 제1 열 블록(100a)과 제2 열 블록(200a) 사이에서 열 교환이 일어나지 않기 때문에, 미세한 온도 변화에 의해서도 중대한 영향을 받을 수 있는 핵산 증폭 반응에 있어서, 상기 변성 단계와 상기 어닐링 및 연장 (혹은 증폭) 단계의 정확한 온도 제어가 가능하다.The first thermal block 100a and the second thermal block 200a may be spaced apart from each other at a predetermined distance such that mutual heat exchange does not occur. Accordingly, since the heat exchange does not occur between the first heat block 100a and the second heat block 200a, in the nucleic acid amplification reaction that may be significantly affected by minute temperature changes, the denaturation step and the Accurate temperature control of the annealing and extension (or amplification) steps is possible.
본 발명의 제4 실시예에 따른 PCR 장치는 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 위로 구동 수단(500a)에 의해 좌우 및/또는 상하 이동 가능하고, PCR 칩(10)이 장착된 칩 홀더(300a)를 포함한다. 상기 칩 홀더(300a)는 상기 PCR 칩(10)이 상기 PCR 장치에 장착되는 모듈이다. 상기 칩 홀더(300a)의 내벽은 상기 PCR 장치에 의해 핵산 증폭 반응이 수행되는 경우 상기 PCR 칩(10)이 상기 칩 홀더(300a)로부터 이탈하지 않도록 상기 PCR 칩(10)의 외벽과 고정 장착되기 위한 형상 및 구조를 가질 수 있다. 상기 칩 홀더(300a)는 상기 구동 수단(500a)에 구동가능하게 연결된다. 또한, 상기 PCR 칩(10)은 상기 칩 홀더(300a)에 착탈 가능할 수 있다.The PCR device according to the fourth embodiment of the present invention may move left and right and / or up and down by the driving means 500a on the first row block 100a and the second row block 200a, and the PCR chip 10 may be used. The mounted chip holder 300a is included. The chip holder 300a is a module in which the PCR chip 10 is mounted to the PCR device. The inner wall of the chip holder 300a is fixedly mounted to the outer wall of the PCR chip 10 so that the PCR chip 10 is not separated from the chip holder 300a when the nucleic acid amplification reaction is performed by the PCR apparatus. It may have a shape and structure for. The chip holder 300a is operably connected to the driving means 500a. In addition, the PCR chip 10 may be detachable to the chip holder (300a).
상기 구동 수단(500a)은 상기 PCR 칩(10)이 장착된 칩 홀더(300a)를 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 위로 좌우 및/또는 상하 이동 가능하게 하는 모든 수단을 포함한다. 상기 구동 수단(500a)의 좌우 이동에 의해, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)는 상기 제1 열 블록(100a)과 제2 열 블록(200a) 사이에서 왕복 운동이 가능하고, 상기 구동 수단(500a)의 상하 이동에 의해, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)는 상기 제1 열 블록(100a)과 제2 열 블록(200a)에 접촉 및 분리될 수 있다. 도 3a에 도시된 본 발명의 제3 실시예에 따른 PCR 장치의 구동 수단(500a)은 좌우 방향으로 연장된 레일(510a), 및 상기 레일(510a)을 통해 좌우 방향으로 슬라이딩 이동가능하게 배치되고, 상하 방향으로 슬라이딩 이동 가능한 연결 부재(520a)를 포함하고, 상기 연결 부재(520a)의 일 말단은 상기 칩 홀더가 배치된다. 상기 구동 수단(500a)의 좌우 및/또는 상하 이동은 상기 PCR 장치의 내부 또는 외부에 구동가능하게 배치된 제어 수단(도시되지 않음)에 의해 제어될 수 있고, 상기 제어 수단은 PCR의 변성 단계와 어닐링 및 연장 (혹은 증폭) 단계를 위한 상기 PCR 칩(10)이 장착된 칩 홀더(300a)와 상기 제1 열 블록(100a) 및 제2 열 블록(200a) 사이의 접촉 및 분리를 제어할 수 있다.The driving means 500a is any means for allowing the chip holder 300a on which the PCR chip 10 is mounted to move left and right and / or up and down over the first row block 100a and the second row block 200a. It includes. By the left and right movement of the driving means 500a, the chip holder 300a on which the PCR chip 10 is mounted is capable of reciprocating between the first row block 100a and the second row block 200a. By the vertical movement of the driving means 500a, the chip holder 300a on which the PCR chip 10 is mounted may contact and be separated from the first row block 100a and the second row block 200a. have. The driving means 500a of the PCR apparatus according to the third embodiment of the present invention shown in FIG. 3A is disposed to be slidably movable in the left and right directions through a rail 510a extending in the left and right directions and the rail 510a. And a connecting member 520a slidable in an up and down direction, and one end of the connecting member 520a is disposed with the chip holder. The left and right and / or vertical movement of the driving means 500a may be controlled by a control means (not shown), which is operably disposed inside or outside the PCR device, and the control means may be modified with a modification step of PCR. It is possible to control the contact and separation between the chip holder 300a on which the PCR chip 10 is mounted and the first row block 100a and the second row block 200a for annealing and extending (or amplifying) the step. have.
도 3b는 본 발명의 제4 실시예에 따른 PCR 장치의 칩 홀더의 이동에 의한 핵산 증폭 반응의 각 단계를 도시한다. 상기 PCR 장치에 의한 핵산 증폭 반응은 하기 단계에 의한다. 먼저, 상기 PCR 칩(10)에 핵산, 예를 들어 이중 가닥 DNA, 증폭하고자 하는 특정 염기 서열과 상보적인 서열을 갖는 올리고뉴클레오티드 프라이머, DNA 중합효소, 삼인산화데옥시리보뉴클레오티드(deoxyribonucleotide triphosphates, dNTP), PCR 완충액(PCR buffer)를 포함하는 샘플 용액을 도입하고, 상기 PCR 칩(10)을 상기 칩 홀더(300a)에 장착하는 단계를 수행한다. 그 후 또는 이와 동시에 상기 제1 열 블록(100a)을 변성 단계를 위한 온도, 예를 들어, 90℃ 내지 100℃로 가열 및 유지하고, 바람직하게는 95℃로 가열 및 유지하는 단계를 수행한다. 상기 제2 열 블록(200)을 어닐링 및 연장 (혹은 증폭) 단계를 위한 온도, 예를 들어, 55℃ 내지 75℃로 가열 및 유지하고, 바람직하게는 72℃로 가열 및 유지하는 단계를 수행한다. 그 후, 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 하향 이동시켜, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)를 상기 제1 열 블록(100a)에 접촉시켜 PCR의 제1 변성 단계를 수행한다(x 단계). 그 후, 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 상향 이동시켜, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)를 상기 제1 열 블록(100a)으로부터 분리시켜 PCR의 제1 변성 단계를 종료하고, 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 제2 열 블록(200a)의 위로 이동시키는 단계를 수행한다(y 단계). 그 후, 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 하향 이동시켜, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)를 상기 제2 열 블록(100a)에 접촉시켜 PCR의 제1 어닐링 및 연장 (혹은 증폭) 단계를 수행한다(z 단계). 마지막으로, 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 상향 이동시켜, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)를 상기 제2 열 블록(100a)으로부터 분리시켜 PCR의 제1 어닐링 및 연장 (혹은 증폭) 단계를 종료하고, 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 제1 열 블록(100a)의 위로 이동시킨 후 상기 x, y, z 단계를 반복함으로써, 핵산 증폭 반응을 수행한다(순환 단계).Figure 3b shows each step of the nucleic acid amplification reaction by the movement of the chip holder of the PCR device according to the fourth embodiment of the present invention. The nucleic acid amplification reaction by the PCR device is based on the following steps. First, a nucleic acid, such as double-stranded DNA, oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, DNA polymerase, deoxyribonucleotide triphosphates (dNTP) in the PCR chip 10. A sample solution including a PCR buffer is introduced, and the PCR chip 10 is mounted on the chip holder 300a. Thereafter or at the same time, the first heat block 100a is heated and maintained at a temperature for the modification step, for example, 90 ° C. to 100 ° C., preferably at 95 ° C. Heating and maintaining the second thermal block 200 at a temperature for annealing and extending (or amplifying), for example, 55 ° C. to 75 ° C., preferably at 72 ° C. . Subsequently, the PCR chip 10 is moved downward by controlling the connecting member 520a of the driving means 500a to move the chip holder 300a on which the PCR chip 10 is mounted to the first row block. 100a) to perform the first denaturation step of PCR (step x). Thereafter, the PCR chip 10 is moved upward by controlling the connecting member 520a of the driving means 500a, so that the chip holder 300a on which the PCR chip 10 is mounted is moved to the first row block ( Separating from 100a) to end the first denaturation step of PCR, and controlling the connecting member 520a of the driving means 500a to move the PCR chip 10 above the second row block 200a. Do it (step y). Subsequently, the PCR chip 10 is moved downward by controlling the connecting member 520a of the driving means 500a to move the chip holder 300a on which the PCR chip 10 is mounted to the second row block ( 100a) to perform the first annealing and extension (or amplification) step of the PCR (step z). Lastly, the PCR chip 10 is moved upward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a on which the PCR chip 10 is mounted is moved to the second row block ( 100a) to terminate the first annealing and extension (or amplification) step of the PCR, and control the connecting member 520a of the driving means 500a to control the PCR chip 10 by the first row block 100a. The nucleic acid amplification reaction is performed by repeating the steps x, y, and z after moving up to (circulation step).
도 3c은 본 발명의 제4 실시예에 따른 PCR 장치를 이용하여 실시간으로 핵산 증폭 반응을 관찰하는 단계를 도시한다. 본 발명의 제3 실시예에 따른 PCR 장치는 상기 제1 열 블록(100a)과 제2 열 블록(200a) 사이에 광원(700a)이 더 배치되고, 상기 칩 홀더(300a) 위에 상기 광원(700a)으로부터 방출되는 광을 검출하기 위한 광 검출부(800a)가 더 배치되거나, 또는 상기 제1 열 블록(100a)과 제2 열 블록(200a) 사이에 광원(700a)으로부터 방출되는 광을 검출하기 위한 광 검출부(800a)가 더 배치되고, 상기 칩 홀더(300a) 위에 광원(700a)이 더 배치될 수 있다. 또한, 상기 광 검출부(800a)는 상기 구동 수단(500a) 위에 배치되고, 상기 구동 수단(900a)은 상기 광원(700a)으로부터 방출되는 광을 통과시키기 위한 관통부(530a)가 배치될 수 있다. 또한, 상기 PCR 칩(10)은 광투과성 재질, 구체적으로 광투과성 플라스틱 재질일 수 있다.Figure 3c shows the step of observing the nucleic acid amplification reaction in real time using a PCR device according to a fourth embodiment of the present invention. In the PCR device according to the third embodiment of the present invention, a light source 700a is further disposed between the first row block 100a and the second row block 200a, and the light source 700a is disposed on the chip holder 300a. An optical detection unit 800a for detecting light emitted from the light emitting device is further disposed, or between the first thermal block 100a and the second thermal block 200a for detecting the light emitted from the light source 700a. The light detector 800a may be further disposed, and the light source 700a may be further disposed on the chip holder 300a. In addition, the light detector 800a may be disposed on the driving means 500a, and the through means 530a may be disposed in the driving means 900a to allow the light emitted from the light source 700a to pass therethrough. In addition, the PCR chip 10 may be a light transmissive material, specifically, a light transmissive plastic material.
상기 광원(700a) 및 광 검출부(800a)의 배치에 의해, 상기 PCR 장치(1)에 의한 핵산 증폭 반응시 상기 PCR 칩(10) 내에서 핵산이 증폭되는 정도를 실시간으로 검출할 수 있도록 한다. 상기 PCR 칩(10) 내에서 핵산이 증폭되는 정도를 검출하기 위해서는 상기 PCR 칩(10)에 도입되는 샘플 용액에 별도의 형광 물질을 더 첨가할 수 있다. 상기 광원(700a)은 상기 제1 열 블록(100a)과 제2 열 블록(200a) 사이의 이격된 공간에 가능한 넓게 분포하도록 배치되고, 가능한 동일한 광을 방출하도록 배치된다. 상기 광원(700a)은 상기 광원(700a)으로부터 방출되는 광을 포집하는 렌즈(도시되지 않음) 및 특정 파장대의 광을 여과하는 광 필터(도시되지 않음)와 구동가능하게 연결 배치될 수 있다.By arranging the light source 700a and the light detector 800a, the nucleic acid amplification reaction can be detected in real time in the PCR chip 10 during the nucleic acid amplification reaction by the PCR apparatus 1. In order to detect the degree of nucleic acid amplification in the PCR chip 10, a separate fluorescent substance may be further added to the sample solution introduced into the PCR chip 10. The light source 700a is arranged to be distributed as widely as possible in the spaced space between the first column block 100a and the second column block 200a, and to emit the same light as much as possible. The light source 700a may be operably connected to a lens (not shown) for collecting light emitted from the light source 700a and an optical filter (not shown) for filtering light of a specific wavelength band.
본 발명의 제4 실시예에 따른 PCR 장치에 의한 핵산 증폭 반응시 상기 PCR 칩(10) 내에서 핵산이 증폭되는 정도를 실시간으로 검출하는 단계는 하기 단계에 의한다. 상기 PCR의 제1 변성 단계의 종료 후 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 제1 열 블록(100a)의 위로부터 제2 열 블록(200a)의 위로 이동시키거나, 또는 상기 PCR의 제1 어닐링 및 연장 (혹은 증폭) 단계의 종료 후 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 PCR 칩(10)을 제2 열 블록(200a)의 위로부터 제1 열 블록(200a)의 위로 이동시키는 경우, 상기 PCR 칩(10)이 장착된 칩 홀더(300a)를 상기 구동 수단(500a)의 연결 부재(520a)를 제어하여 상기 제1 열 블록(100a)과 제2 열 블록(200a) 사이의 이격된 공간 상에 정지시키는 단계를 수행한다. 그 후, 상기 광원(700a)으로부터 광을 방출시키고, 상기 방출된 광은 상기 광투과성 PCR 칩(10), 구체적으로 상기 PCR 칩(10)의 반응 채널)를 통과하고, 이 경우 상기 반응 채널) 내의 핵산의 증폭에 의해 발생하는 광 신호를 상기 광 검출부(800a)가 검출한다. 이 경우 상기 광투과성 PCR 칩(10)을 통과한 광은 상기 구동 수단(500a), 구체적으로 상기 레일(510a)에 배치된 관통부(530a)를 통과하여 상기 광출부(800a)에 도달할 수 있다. 따라서, 본 발명의 제3 실시예에 따른 PCR 장치에 따르면, 상기 PCR의 각 순환 단계가 진행되는 동안 상기 반응 채널 내에서 (형광 물질이 결합된) 핵산의 증폭에 의한 반응 결과를 실시간으로 모니터링함으로써 초기 반응 샘플에 포함되어 있는 표적 핵산의 양을 실시간으로 측정 및 분석할 수 있다.In the nucleic acid amplification reaction by the PCR device according to the fourth embodiment of the present invention, the step of detecting in real time the degree of nucleic acid amplification in the PCR chip 10 is based on the following steps. After completion of the first denaturation step of the PCR, the connection member 520a of the driving means 500a is controlled to move the PCR chip 10 from above the first row block 100a to the second row block 200a. After moving up or after the first annealing and extending (or amplifying) step of the PCR, the connecting member 520a of the driving means 500a is controlled to move the PCR chip 10 to the second row block 200a. When moving from above the first row block 200a to the first row block 200a, the chip holder 300a on which the PCR chip 10 is mounted is controlled to control the connecting member 520a of the driving means 500a. A step of stopping on the spaced space between the row block 100a and the second row block 200a is performed. Thereafter, light is emitted from the light source 700a, and the emitted light passes through the light transmissive PCR chip 10, specifically, the reaction channel of the PCR chip 10, in which case the reaction channel) The light detector 800a detects an optical signal generated by amplification of the nucleic acid therein. In this case, the light passing through the light transmissive PCR chip 10 may reach the light exit portion 800a by passing through the driving means 500a, specifically, the through portion 530a disposed on the rail 510a. have. Therefore, according to the PCR device according to the third embodiment of the present invention, by monitoring the reaction result by the amplification of nucleic acid (phosphorescent material bound) in the reaction channel in real time during each cyclic step of the PCR by The amount of target nucleic acid included in the initial reaction sample can be measured and analyzed in real time.
도 4a 내지 도 4i는 본 발명의 제5 실시예에 따른 PCR 장치에 관한 도면이다.4A to 4I are diagrams illustrating a PCR device according to a fifth embodiment of the present invention.
도 4a는 본 발명의 제5 실시예에 따른 PCR 장치에 포함된 광 투과성 열 블록(100b)을 도시한다. 상기 PCR 장치는 기판(10b), 상기 기판(10b) 상에 배치된 도전성 나노 입자를 포함하는 발열층(20b), 상기 발열층 상에 배치된 절연 보호층(30b) 및 상기 발열층과 연결 배치된 전극(40b)을 구비하되, 광 투과성을 갖도록 구현된 광투과성 열 블록(100b); 및 상기 광투과성 열 블록(100b)의 상부 면에 접촉 가능하도록 배치된, 본 발명의 제3 구체예에 따른 PCR 칩(10)을 포함한다.4A shows a light transmitting thermal block 100b included in a PCR device according to a fifth embodiment of the present invention. The PCR device includes a substrate 10b, a heating layer 20b including conductive nanoparticles disposed on the substrate 10b, an insulating protective layer 30b disposed on the heating layer, and a connection arrangement with the heating layer. A light-transmitting thermal block 100b having an electrode 40b formed thereon, the light-transmitting thermal block 100b being implemented to have light transmission; And a PCR chip 10 according to the third embodiment of the present invention, disposed to be in contact with the upper surface of the light transmissive thermal block 100b.
상기 기판(10b)은 광 투과성 재질의 판재로서, 광 투과성 유리 또는 광 투과성 플라스틱 재질일 수 있다. 상기 발열층(20b)은 PCR의 변성 단계, 어닐링 단계 및 연장(또는 증폭) 단계를 수행하기 위한 상기 광 투과성 열 블록(100b)의 열원 역할을 수행한다. 상기 도전성 나노 입자는 산화물 반도체 물질 또는 상기 산화물 반도체 물질에 In, Sb, Al, Ga, C 및 Sn로 구성된 군으로부터 선택된 불순물이 첨가된 물질일 수 있다. 또한, 상기 발열층(20)은 상기 도전성 나노 입자가 물리적으로 연계(necking)된 성긴 조직(loose texture) 구조를 가질 수 있고, 제조 공정의 열 처리 조건에 따라 치밀한 조직(close-packed texture)을 가질 수 있으며, 또한 완전한 막 상태로 구현될 수도 있다. 또한, 상기 도전성 나노 입자는 용매에 분산된 상태로 존재하므로, 상기 기판(10b) 상에 용이하게 적층할 수 있기 때문에, 그 적층 수를 조절하여 상기 발열층(20b)의 두께 조절을 용이하게 할 수 있다. 또한, 상기 도전성 나노 입자를 포함하는 분산액의 농도를 조절함으로써 상기 발열층(20b)의 도전성을 용이하게 조절할 수도 있다. 또한, 상기 발열층(20b)을 상기 기판(10b)에 강하게 고정하기 위하여 상기 기판(10b)과 발열층(20b) 사이에 접착력 강화층(도시되지 않음)이 형성될 수 있다. 상기 접착력 강화층은 실리카 또는 폴리머로 형성될 수 있고, 도전성 나노 입자를 포함할 수 있어 발열층과 동일한 역할을 또한 수행할 수도 있다. 또한, 상기 발열층(20b)은 투명할 수 있다. 예를 들어, 가시광선의 파장은 400 내지 700 nm이고, 도전성 나노 입자를 포함하는 발열층을 이러한 파장의 1/4 이하의 두께, 예를 들어 약 100 nm 이하가 되도록 형성하는 경우 광 투과성을 획득할 수 있다. 상기 절연 보호층(30b)은 상기 발열층(20b)을 물리적 및/또는 전기적으로 보호하기 위한 것으로서, 절연성 물질을 포함할 수 있다. 예를 들어, 상기 절연성 물질은 유전체 산화물, 페릴린, 나노 입자 및 고분자 필름으로 구성된 군으로부터 선택될 수 있다. 한편, 상기 절연 보호층(30b)은 투명할 수 있다. 상기 전극(40b)은 상기 발열층(20)과 직접 또는 간접적으로 연결 배치되어 상기 발열층(20b)에 전력을 공급하는 것이다. 상기 전극(40b)은 전력을 공급할 수 있는 다양한 물질이 사용될 수 있고, 예를 들어 금속 물질, 전도성 에폭시, 전도성 페이스트, 솔더 및 전도성 필름으로 구성된 군으로부터 선택될 수 있다. 도 4a에 따르면, 상기 전극(40b)은 상기 발열층(20b)의 양 측면에 연결 배치되지만, 상기 발열층(20b)에 전력을 공급할 수 있다면 다양하게 작동가능한 위치에서 연결 배치될 수 있다. 또한, 상기 전극(40b)은 상기 PCR 장치에 포함되거나 또는 외부 배치된 전원과 전기적으로 연결될 수도 있다. 예를 들어, 상기 전극(40b)은 상기 발열층(20b)에 직접 접촉하고, 배선(도시되지 않음)을 통해 외부 회로(도시되지 않음)에 상기 발열층(20b)을 연결하며, 상기 배선이 전극(40b)에 안정적으로 고정되도록 단자가 배치될 수 있다. The substrate 10b is a plate of light transmissive material, and may be light transmissive glass or light transmissive plastic material. The heating layer 20b serves as a heat source of the light transmitting thermal block 100b for performing the denaturation step, annealing step, and extension (or amplification) step of PCR. The conductive nanoparticle may be an oxide semiconductor material or a material to which an impurity selected from the group consisting of In, Sb, Al, Ga, C, and Sn is added to the oxide semiconductor material. In addition, the heat generating layer 20 may have a loose texture structure in which the conductive nanoparticles are physically linked to each other, and may generate a close-packed texture according to heat treatment conditions of a manufacturing process. It may also have a complete film state. In addition, since the conductive nanoparticles are present in a dispersed state in a solvent, the conductive nanoparticles can be easily stacked on the substrate 10b, so that the thickness of the heat generating layer 20b can be easily adjusted by controlling the number of stacked layers. Can be. In addition, the conductivity of the heating layer 20b may be easily adjusted by adjusting the concentration of the dispersion liquid containing the conductive nanoparticles. In addition, an adhesion strengthening layer (not shown) may be formed between the substrate 10b and the heating layer 20b to strongly fix the heating layer 20b to the substrate 10b. The adhesion reinforcing layer may be formed of silica or a polymer, may include conductive nanoparticles may also play the same role as the heating layer. In addition, the heating layer 20b may be transparent. For example, the wavelength of visible light is 400 to 700 nm, and when a heat generating layer including conductive nanoparticles is formed to have a thickness of 1/4 or less of such wavelength, for example, about 100 nm or less, light transmittance may be obtained. Can be. The insulating protective layer 30b is for physically and / or electrically protecting the heating layer 20b and may include an insulating material. For example, the insulating material may be selected from the group consisting of dielectric oxides, perylenes, nanoparticles, and polymer films. Meanwhile, the insulation protection layer 30b may be transparent. The electrode 40b is connected to the heat generating layer 20 directly or indirectly to supply power to the heat generating layer 20b. Various materials capable of supplying power may be used as the electrode 40b, and may be selected from, for example, a metal material, a conductive epoxy, a conductive paste, a solder, and a conductive film. According to FIG. 4A, the electrodes 40b are connected to both sides of the heating layer 20b, but may be connected at various operable positions if power can be supplied to the heating layer 20b. In addition, the electrode 40b may be included in the PCR device or electrically connected to an externally arranged power source. For example, the electrode 40b directly contacts the heat generating layer 20b, and connects the heat generating layer 20b to an external circuit (not shown) through a wire (not shown). The terminal may be arranged to be stably fixed to the electrode 40b.
상기 광 투과성 열 블록(100b)은 그 상부 면의 적어도 일부 영역에 PCR 칩(도시되지 않음)이 접촉하는 칩 접촉부(50b)를 포함한다. 상기 PCR 칩(10)은 상기 칩 접촉부(50b)에 접촉함으로써, 상기 광 투과성 열 블록(100b)의 열 공급 또는 회수에 따라 가열 또는 냉각되어 PCR의 각 단계를 수행할 수 있다. 또한, 상기 PCR 칩(10)은 상기 칩 접촉부(50)에 직접 또는 간접적으로 접촉할 수 있다. 상기 광 투과성 열 블록(100b)을 포함하는 PCR 장치는 종래 기존 석열 히터, 세라믹 히터 또는 금속 히터를 열 블록으로 이용하는 PCR 장치에 비해 많은 장점을 갖는다. 먼저, 열원으로서 도전성 나노 입자를 이용하기 때문에 발열층의 단선의 우려가 없고, 상기 도전성 나노 입자를 직접적으로 가열하기 때문에 높은 열 효율 및 낮은 소비 전력을 획득할 수 있으며(예를 들어, 상기 광투과성 열 블록이 약 2X2 ㎝의 규격일 경우 약 12V의 전압으로 발열이 가능함), 금속 재질이 아니므로 산화, 부식이 거의 일어나지 않아 내구성이 뛰어나다. 또한, 상기 기판(10b), 발열층(20b) 및 절연 보호층(30b)의 제조시 광 투과성을 획득할 수 있기 때문에, 이하 설명될 광 제공부 및 광 검출부와 함께 구현될 경우 샘플 용액에 포함된 형광 물질에 의한 PCR의 실시간 모니터링이 가능하다. 또한, 상기 기판(10b), 발열층(20b) 및 절연 보호층(30b)의 제조시 그 두께 조절이 용이하기 때문에 상기 광 투과성 열 블록(100b)의 슬림(slim)화가 가능하여 상기 광 투과성 열 블록(100b)을 포함하는 PCR 장치의 소형화가 가능하다. 또한, 상기 도전성 나노 입자가 상기 발열층(20b)에 균일하게 분포되어 상기 광 투과성 열 블록(100b)의 균일한 열 분포 및 신속한 온도 제어가 가능하기 때문에 PCR 결과의 검출 효율이 높고, PCR 결과를 신속하게 얻을 수 있다. 상기 광 투과성 열 블록(100b)의 열 분포의 균일성 및 온도 제어의 신속성은 도 2에 따른 실험 결과로서 확인할 수 있다. 도 4b는 본 발명의 제5 실시예에 따른 PCR 장치에 포함된 광 투과성 열 블록(100b)의 시간에 따른 온도 변화를 도시한다. 기존 PCR 장치에서 열 블록으로 사용된 석열 히터, 세라믹 히터 또는 금속 히터에 전력을 인가하여 온도 분포를 관찰하고, 상기 광 투과성 열 블록(100b)에 상기 전극(40b)을 통해 전력을 인가하여 온도 분포를 관찰하였다. 그 결과, 기존 히터 상의 온도 분포는 히터 표면 전체에 걸쳐서 균일하지 않지만, 상기 광 투과성 열 블록(100b) 상의 온도 분포는 상기 기존 히터에 비해 전체적으로 균일한 것으로 관찰되었다. 또한, 상기 광 투과성 열 블록(100b)에 상기 전극(40b)을 통해 전력을 인가하여 시간에 따른 상기 광 투과성 열 블록(100b)의 온도 변화를 관찰하였다. 그 결과, 온도 상승 폭은 최대 17 ℃/sec으로 나타났고, 이는 대표적인 기존 히터들(예를 들어, Bio-Rad사의 CFX96)의 온도 상승 폭이 최대 5 ℃/sec인 것에 비해 상당히 높은 수치임을 확인할 수 있다.The light transmissive thermal block 100b includes a chip contact 50b to which a PCR chip (not shown) contacts at least a portion of an upper surface thereof. The PCR chip 10 may be heated or cooled according to the heat supply or recovery of the light transmitting thermal block 100b by contacting the chip contact part 50b to perform each step of PCR. In addition, the PCR chip 10 may directly or indirectly contact the chip contact 50. The PCR device including the light transmitting thermal block 100b has many advantages over the conventional PCR device using a conventional heater, ceramic heater, or metal heater as a thermal block. First, since the conductive nanoparticles are used as the heat source, there is no fear of disconnection of the heating layer, and since the conductive nanoparticles are directly heated, high thermal efficiency and low power consumption can be obtained (for example, the light transmittance If the heat block is about 2X2 ㎝ size, it can generate heat with a voltage of about 12V.) Since it is not a metal material, it hardly oxidizes and corrodes, so it has excellent durability. In addition, since the light transmittance may be obtained when the substrate 10b, the heat generating layer 20b, and the insulating protective layer 30b are manufactured, when included together with the light providing unit and the light detecting unit to be described below, the sample 10 may be included in the sample solution. Real-time monitoring of PCR by fluorescent material is possible. In addition, since the thickness of the substrate 10b, the heat generating layer 20b, and the insulating protective layer 30b can be easily adjusted, the light transmitting heat block 100b can be slimmed, thereby allowing the light transmitting heat to be reduced. Miniaturization of the PCR device including the block 100b is possible. In addition, since the conductive nanoparticles are uniformly distributed in the heat generating layer 20b, uniform heat distribution and rapid temperature control of the light transmissive thermal block 100b are possible. You can get it quickly. The uniformity of the heat distribution of the light transmitting thermal block 100b and the rapidity of temperature control can be confirmed as an experimental result according to FIG. 2. 4B illustrates a temperature change with time of the light transmissive thermal block 100b included in the PCR device according to the fifth embodiment of the present invention. The temperature distribution is observed by applying electric power to the calcite heater, the ceramic heater, or the metal heater used as the thermal block in the conventional PCR apparatus, and the temperature distribution by applying electric power to the light transmitting thermal block 100b through the electrode 40b. Was observed. As a result, the temperature distribution on the existing heater was not uniform throughout the heater surface, but the temperature distribution on the light transmissive heat block 100b was observed to be overall uniform compared to the conventional heater. In addition, power was applied to the light transmitting thermal block 100b through the electrode 40b to observe a temperature change of the light transmitting thermal block 100b over time. As a result, the temperature rise was shown to be up to 17 ℃ / sec, which indicates that the temperature rise of the typical conventional heaters (for example, Bio-Rad's CFX96) is significantly higher than the maximum rise of 5 ℃ / sec. Can be.
도 4c는 기판(10b) 하부 면에 흡광층(60b)이 접촉 배치된 본 발명의 제5 실시예에 따른 PCR 장치에 포함된 광 투과성 열 블록(100b)을 도시하고, 도 4d는 절연 보호층(30b) 상부 면에 광반사방지층(70b)이 접촉 배치된 본 발명의 제5 실시예에 따른 PCR 장치에 포함된 광 투과성 열 블록(100b)을 도시하고, 도 4e는 기판(10b) 하부면에 흡광층(60b)이 접촉 배치되고, 외부 공기층과 절연보호층(30b)의 접촉에 의한 광 반사를 방지하기 위한 광반사방지층(70b)이 상기 절연보호층(30b)의 상부에 접촉 배치된 본 발명의 제5 실시예에 따른 PCR 장치에 포함된 광 투과성 열 블록(100b)을 도시한다.FIG. 4C shows a light transmissive thermal block 100b included in a PCR device according to a fifth embodiment of the present invention in which a light absorbing layer 60b is disposed in contact with a bottom surface of a substrate 10b, and FIG. 4D shows an insulating protective layer. 30b illustrates a light transmissive thermal block 100b included in a PCR device according to a fifth embodiment of the present invention in which a light reflection prevention layer 70b is disposed in contact with an upper surface thereof, and FIG. 4e illustrates a bottom surface of the substrate 10b. The light absorbing layer 60b is disposed in contact with the light absorbing layer 60b, and the light reflection preventing layer 70b for preventing light reflection due to the contact between the external air layer and the insulating protective layer 30b is in contact with the upper portion of the insulating protective layer 30b. A light transmissive thermal block 100b included in a PCR device according to a fifth embodiment of the present invention is shown.
일반적으로, PCR을 수행함과 동시에 형광물질을 이용하여 PCR 산물의 발생 유무 및 정도를 실시간으로 측정 및 분석할 수 있다. 이와 같은 PCR을 소위 실시간 중합효소 연쇄반응(Real time PCR)이라고 한다. 상기 반응은 PCR 칩에 PCR 반응에 필요한 시약뿐만 아니라 형광물질이 첨가되고, PCR 산물의 생성에 따라 상기 형광물질이 특정 파장의 광에 의해 발광함으로써 측정 및 분석 가능한 광 신호를 유발하게 된다. 따라서, 실시간으로 PCR 산물을 정확하게 모니터링하기 위해서는 상기 광 신호의 센싱 효율을 가능한 높힐 필요가 있다. 상기 광 투과성 열 블록(100b)은 전체적으로 광 투과성을 갖기 때문에 광원으로부터 유래된 여기 광을 대부분 그대로 투과시켜 상기 광 신호의 센싱 효율을 높힐 수 있다. 그러나, 상기 여기 광의 일부는 상기 광 투과성 열 블록(100b) 상에서 반사되거나 또는 상기 광 투과성 열 블록(100b)을 통과한 후 반사되어 광 신호의 노이즈(noise)로서 작용할 수 있다. 따라서, 바람직하게는, 상기 광 투과성 열 블록(100b)의 하부 면에 흡광 물질을 처리하여 센싱 효율을 더욱 높힐 수 있다. 도 4c에 따르면, 흡광층(60b)이 상기 기판(10b)의 하부 면에 접촉 배치되고, 상기 흡광층(60b)은 흡광 물질을 포함한다. 상기 흡광 물질은 예를 들어, 운모(mica)일 수 있으나, 광을 흡수하는 성질을 갖는 물질이라면 제한되지 않는다. 따라서, 광원으로부터 유래된 광의 일부를 상기 흡광층(60b)이 흡수하여, 광 신호의 노이즈로 작용하는 반사 광의 발생을 최대한 억제할 수 있다. 또한, 대안적으로, 상기 광 투과성 열 블록(100b)의 상부 면에 광반사방지 물질을 처리하여 센싱 효율을 더욱 높힐 수 있다. 도 4d에 따르면, 광반사방지층(70b)이 상기 절연 보호층(30b)의 상부 면에 접촉 배치되고, 상기 광반사방지층(70b)은 절연보호층 (30b)과 조합하여 절연보호 기능 및 광반사방지 기능을 수행하며, 광반사방지 물질을 포함한다. 상기 광반사방지 물질은 예를 들어, MgF2와 같은 불화물, SiO2, Al2O3와 같은 산화물일 수 있으나, 광반사를 방지할 수 있는 성질을 갖는 물질이라면 제한되지 않는다. 또한, 더 바람직하게는, 상기 광 투과성 열 블록(100b)의 하부 면에 흡광 물질을 처리하고, 동시에 상기 광 투과성 열 블록(100b)의 상부 면에 광반사방지 물질을 처리하여 센싱 효율을 더욱 높힐 수 있다. 즉, 효과적인 실시간 PCR의 모니터링을 위하여 상기 노이즈 대비 광 신호 비율은 가능한 최대값을 가져야 하고, 상기 노이즈 대비 광 신호 비율은 상기 PCR 칩으로부터 여기 광의 반사율이 낮을수록 향상될 수 있다. 예를 들어, 일반적인 금속성 재질의 기존 히터들의 여기 광의 반사율은 약 20 내지 80 %이지만, 상기 도 4c 또는 도 4d에 따른 흡광층(60b) 또는 광반사방지층(70b)을 포함하는 광 투과성 열 블록(100b)을 사용하는 경우 광 반사율을 0.2% 내지 4% 이내로 줄일 수 있고, 상기 도 4e에 따른 흡광층(60b) 및 광반사방지층(70b)을 포함하는 본 발명에 따른 광 투과성 열 블록(100b)을 사용하는 경우 광 반사율을 0.2% 이하로 줄일 수 있다.In general, it is possible to measure and analyze the occurrence and extent of PCR products in real time using a fluorescent material while performing a PCR. Such PCR is called a real time polymerase chain reaction (Real time PCR). In the reaction, a fluorescent material as well as a reagent required for a PCR reaction is added to a PCR chip, and the fluorescent material emits light by light of a specific wavelength according to the generation of a PCR product, thereby inducing a measurable optical signal. Therefore, in order to accurately monitor the PCR product in real time, it is necessary to increase the sensing efficiency of the optical signal as much as possible. Since the light transmitting thermal block 100b has light transmittance as a whole, the excitation light derived from the light source may be transmitted as it is, thereby increasing the sensing efficiency of the optical signal. However, some of the excitation light may be reflected on the light transmissive heat block 100b or reflected after passing through the light transmissive heat block 100b to act as noise of an optical signal. Therefore, preferably, the light absorbing material may be treated on the lower surface of the light transmitting thermal block 100b to further increase the sensing efficiency. According to FIG. 4C, the light absorbing layer 60b is disposed in contact with the lower surface of the substrate 10b, and the light absorbing layer 60b includes a light absorbing material. The light absorbing material may be, for example, mica, but is not limited to a material having a property of absorbing light. Therefore, the light absorption layer 60b absorbs a part of the light derived from the light source, and the generation of reflected light acting as noise of the optical signal can be suppressed as much as possible. Alternatively, the light reflection preventing material may be treated on the upper surface of the light transmissive thermal block 100b to further increase the sensing efficiency. According to FIG. 4D, the light reflection prevention layer 70b is disposed in contact with the upper surface of the insulating protection layer 30b, and the light reflection prevention layer 70b is combined with the insulation protection layer 30b to provide insulation protection and light reflection. Performs a protective function and includes an antireflective material. The anti-reflective material may be, for example, a fluoride such as MgF 2, an oxide such as SiO 2 or Al 2 O 3, but is not limited as long as the material has a property of preventing light reflection. Further, more preferably, the light absorbing material is treated on the lower surface of the light transmitting thermal block 100b, and at the same time, the light reflection preventing material is treated on the upper surface of the light transmitting thermal block 100b to further increase the sensing efficiency. Can be. That is, for effective real-time PCR monitoring, the ratio of the optical signal to the noise should have the maximum possible value, and the ratio of the optical signal to the noise may be improved as the reflectance of the excitation light from the PCR chip is lower. For example, although the reflectance of the excitation light of conventional heaters of a general metallic material is about 20 to 80%, the light transmitting thermal block including the light absorbing layer 60b or the antireflective layer 70b according to FIG. 4C or 4D ( In the case of using 100b), the light reflectance can be reduced to within 0.2% to 4%, and the light transmitting thermal block 100b according to the present invention includes a light absorbing layer 60b and an antireflective layer 70b according to FIG. 4E. When using the light reflectance can be reduced to 0.2% or less.
도 4f는 광 제공부 및 광 검출부를 포함하는 본 발명의 제5 실시예에 따른 PCR 장치의 광 투과성 열 블록(100b) 상에 PCR 칩(10)이 배치된 것을 도시한다. 도 4f에 따르면, 상기 PCR 장치는 상기 칩 접촉부(50b)에 배치되는 PCR 칩(10)에 광을 제공하도록 구동가능하게 배치된 광 제공부(200b) 및 상기 칩 접촉부(50b)에 배치되는 PCR 칩(10)으로부터 방출되는 광을 수용하도록 구동가능하게 배치된 광 검출부(300b)를 더 포함한다. 상기 광 제공부(200b)는 상기 PCR 칩(10)에 광을 제공하기 위한 모듈이고, 상기 광 검출부(300b)는 상기 PCR 칩(10)으로부터 방출되는 광을 수용하여 상기 PCR 칩(10)에서 수행되는 PCR 반응을 측정하기 위한 모듈이다. 상기 광 제공부(200b)로부터 광이 방출되고, 상기 방출된 광은 상기 PCR 칩(10), 구체적으로 상기 PCR 칩(10)의 반응 채널(도시되지 않음)을 통과하거나 반사하고, 이 경우 상기 반응 채널 내의 핵산 증폭에 의해 발생하는 광 신호를 상기 광 검출부(300b)가 검출한다. 따라서, 본 발명의 제5 실시예에 따른 PCR 장치에 따르면, 상기 PCR 칩(10)에서 상기 PCR의 각 순환 단계가 진행되는 동안 상기 반응 채널 내에서 (형광 물질이 결합된) 핵산의 증폭에 의한 반응 결과를 실시간으로 모니터링함으로써 초기 샘플 용액에 포함되어 있는 표적 핵산의 증폭 여부 및 증폭 정도를 실시간으로 측정 및 분석할 수 있다. 또한, 상기 광 제공부(200b) 및 광 검출부(300b)는 상기 광 투과성 열 블록(100b)을 중심으로 위 또는 아래에 모두 배치되거나 각각 배치될 수 있다. 다만, 상기 광 제공부(200b) 및 광 검출부(300b)의 배치는 최적의 구현을 위하여 다른 모듈과의 배치 관계를 고려하여 다양할 수 있으며, 바람직하게는 도 4f에 따라, 상기 광 제공부(200b) 및 광 검출부(300b)은 상기 광 투과성 열 블록의 상부에 배치될 수 있다.4F shows that the PCR chip 10 is disposed on the light transmissive heat block 100b of the PCR device according to the fifth embodiment of the present invention including the light providing unit and the light detecting unit. According to FIG. 4F, the PCR device includes a light providing unit 200b disposed to be operable to provide light to the PCR chip 10 disposed on the chip contact unit 50b and a PCR disposed on the chip contact unit 50b. The apparatus further includes a light detector 300b operably disposed to receive light emitted from the chip 10. The light providing unit 200b is a module for providing light to the PCR chip 10, and the light detecting unit 300b receives the light emitted from the PCR chip 10 in the PCR chip 10. Module for measuring the PCR reaction performed. Light is emitted from the light providing unit 200b, and the emitted light passes or reflects through the PCR chip 10, specifically, a reaction channel (not shown) of the PCR chip 10, in which case the The light detector 300b detects an optical signal generated by nucleic acid amplification in the reaction channel. Therefore, according to the PCR device according to the fifth embodiment of the present invention, the amplification of nucleic acid (phosphorescent material bound) in the reaction channel during each cyclic step of the PCR in the PCR chip 10 by By monitoring the result of the reaction in real time, it is possible to measure and analyze in real time whether the target nucleic acid contained in the initial sample solution and the degree of amplification. In addition, the light providing unit 200b and the light detecting unit 300b may be disposed above or below the light transmitting thermal block 100b, or may be disposed respectively. However, the arrangement of the light providing unit 200b and the light detecting unit 300b may vary in consideration of the arrangement relationship with other modules for optimal implementation. Preferably, the light providing unit ( 200b) and the light detector 300b may be disposed on the light transmitting thermal block.
한편, 도 4d에 따르면, 상기 광 제공부(200b)는 LED(Light Emitting Diode) 광원 또는 레이저 광원(210b), 상기 광원으로부터 방출되는 광에서 미리 결정된 파장을 갖는 광을 선택하는 제1 광 여과기(230b), 및 상기 제1 광 여과기로부터 방출되는 광을 포집하는 제1 광 렌즈(240b)를 포함하고, 상기 광원(210b)과 상기 제1 광 여과기(230b) 사이에 빛을 퍼지게 하도록 배치된 제1 비구면 렌즈(220b)를 더 포함한다. 상기 광원(210b)은 광을 방출할 수 있는 모든 광원을 포함하며, LED(Light Emitting Diode) 광원 또는 레이저 광원을 포함한다. 상기 제1 광 여과기(230b)는 다양한 파장대를 갖는 입사광 중 특정 파장의 광을 선택하여 방출하는 것으로, 미리 결정된 상기 광원(210b)에 따라 다양하게 선택될 수 있다. 예를 들어, 상기 제1 광 여과기(230b)는 상기 광원(210b)으로부터 방출되는 광 중 500 nm 이하 파장대의 광만을 통과시킬 수 있다. 상기 제1 광 렌즈(240b)는 그 입사광을 포집하여 그 방출광의 강도를 증가시키는 역할을 수행하는 것으로, 상기 광 투과성 열 블록(100b)을 통해 PCR 칩(10)에 조사되는 광의 강도를 증가시킬 수 있다. 또한, 상기 광 제공부(200b)은 상기 광원(210b)과 상기 제1 광 여과기(230b) 사이에 빛을 퍼지게 하도록 배치된 제1 비구면 렌즈(220b)를 더 포함한다. 상기 제1 비구면 렌즈(220b)의 배치 방향을 조정함으로써, 상기 광원(210b)으로부터 방출되는 광 범위를 확장하여 측정 가능한 영역에 도달하게 한다.Meanwhile, according to FIG. 4D, the light providing unit 200b may include a light emitting diode (LED) light source or a laser light source 210b and a first light filter for selecting light having a predetermined wavelength from light emitted from the light source. 230b) and a first optical lens 240b for collecting light emitted from the first light filter, the first optical lens 240b being arranged to spread light between the light source 210b and the first light filter 230b. It further includes one aspherical lens (220b). The light source 210b includes all light sources capable of emitting light, and includes a light emitting diode (LED) light source or a laser light source. The first light filter 230b selects and emits light having a specific wavelength among incident light having various wavelength bands, and may be variously selected according to the predetermined light source 210b. For example, the first light filter 230b may pass only light in a wavelength band of 500 nm or less among the light emitted from the light source 210b. The first optical lens 240b collects the incident light and increases the intensity of the emitted light. The first optical lens 240b increases the intensity of light irradiated onto the PCR chip 10 through the light transmitting thermal block 100b. Can be. In addition, the light providing unit 200b further includes a first aspherical lens 220b disposed to spread light between the light source 210b and the first light filter 230b. By adjusting the arrangement direction of the first aspherical lens 220b, the light range emitted from the light source 210b is extended to reach the measurable area.
도 4h에 따르면, 상기 광 검출부(300b)는 상기 칩 접촉부(50b)에 배치되는 PCR 칩(10)으로부터 방출되는 광을 포집하는 제2 광 렌즈(310b), 상기 제2 광 렌즈로부터 방출되는 광에서 미리 결정된 파장을 갖는 광을 선택하는 제2 광 여과기(320b), 및 상기 제2 광 여과기(320b)로부터 방출되는 광으로부터 광 신호를 검출하는 광 분석기(350b)를 포함하고, 상기 제2 광 여과기(320b)와 상기 광 분석기(350b) 사이에 상기 제2 광 여과기(320b)로부터 방출되는 광을 집적하도록 배치된 제2 비구면 렌즈(330b)를 더 포함하며, 상기 제2 비구면 렌즈(330b)와 상기 광 분석기(350b) 사이에 상기 제2 비구면 렌즈(330b)로부터 방출되는 광의 노이즈(noise)를 제거하고 상기 제2 비구면 렌즈로(330b)부터 방출되는 광을 증폭하도록 배치된 광다이오드 집적소자(photodiode integrated circuit, PDIC)(340b)를 더 포함한다. 상기 제2 광 렌즈(310b)는 그 입사광을 포집하여 그 방출광의 강도를 증가시키는 역할을 수행하는 것으로, 상기 광 투과성 열 블록(100b)을 통해 PCR 칩(10)으로부터 방출되는 광의 강도를 증가시켜 광 신호 검출을 용이하게 한다. 상기 제2 광 여과기(320b)는 다양한 파장대를 갖는 입사광 중 특정 파장의 광을 선택하여 방출하는 것으로, 상기 광 투과성 열 블록(100b)을 통해 PCR 칩(10)으로부터 방출되는 미리 결정된 광의 파장에 따라 다양하게 선택될 수 있다. 예를 들어, 상기 제2 광 여과기(320b)는 상기 광 투과성 열 블록(100b)을 통해 PCR 칩(10)으로부터 방출되는 미리 결정된 광 중 500 nm 이하 파장대의 광만을 통과시킬 수 있다. 상기 광 분석기(350b)는 상기 제2 광 여과기(320b)로부터 방출되는 광으로부터 광 신호를 검출하는 모듈로서, 샘플 용액으로부터 발현 형광된 광을 전기 신호로 전환하여 정성 및 정략적인 측정이 가능하도록 한다. 또한, 상기 광 검출부(300b)는 상기 제2 광 여과기(320b)와 상기 광 분석기(350b) 사이에 상기 제2 광 여과기(320b)로부터 방출되는 광을 집적하도록 배치된 제2 비구면 렌즈(330b)를 더 포함할 수 있다. 상기 제2 비구면 렌즈(330b)의 배치 방향을 조정함으로써, 상기 제2 광 여과기(320b)로부터 방출되는 광 범위를 확장하여 측정 가능한 영역에 도달하게 한다. 또한, 상기 광 검출부(300b)는 상기 제2 비구면 렌즈(330b)와 상기 광 분석기(350b) 사이에 상기 제2 비구면 렌즈(330b)로부터 방출되는 광의 노이즈(noise)를 제거하고, 상기 제2 비구면 렌즈(330b)로부터 방출되는 광을 증폭하도록 배치된 광다이오드 집적소자(photodiode integrated circuit, PDIC)(340b)를 더 포함할 수 있다. 상기 광다이오드 집적소자(340b)를 사용함으로써, PCR 장치의 소형화가 더욱 가능하고, 노이즈를 최소화하여 신뢰 가능한 광 신호를 측정할 수 있다.According to FIG. 4H, the light detector 300b includes a second optical lens 310b for collecting light emitted from the PCR chip 10 disposed on the chip contact 50b, and the light emitted from the second optical lens. A second light filter 320b for selecting light having a predetermined wavelength at and an optical analyzer 350b for detecting an optical signal from the light emitted from the second light filter 320b, wherein the second light filter And further comprising a second aspherical lens 330b disposed between the filter 320b and the optical analyzer 350b to integrate light emitted from the second light filter 320b, wherein the second aspherical lens 330b is provided. And an optical diode integrated device disposed between the optical analyzer 350b to remove noise of light emitted from the second aspherical lens 330b and to amplify the light emitted from the second aspherical lens 330b. (photodiode integrated circuit, PDIC) (340b) more The. The second optical lens 310b collects the incident light and increases the intensity of the emitted light. The second optical lens 310b increases the intensity of light emitted from the PCR chip 10 through the light transmitting thermal block 100b. Facilitate optical signal detection. The second light filter 320b selects and emits light having a specific wavelength among incident light having various wavelength bands, and is determined according to a wavelength of predetermined light emitted from the PCR chip 10 through the light transmitting thermal block 100b. Various choices can be made. For example, the second light filter 320b may pass only light in a wavelength band of 500 nm or less among predetermined light emitted from the PCR chip 10 through the light transmitting thermal block 100b. The optical analyzer 350b is a module that detects an optical signal from light emitted from the second optical filter 320b. The optical analyzer 350b converts light fluorescence expressed from a sample solution into an electrical signal to enable qualitative and quantitative measurement. . In addition, the light detector 300b may include a second aspheric lens 330b disposed between the second light filter 320b and the light analyzer 350b to integrate light emitted from the second light filter 320b. It may further include. By adjusting the arrangement direction of the second aspherical lens 330b, the light range emitted from the second light filter 320b is extended to reach the measurable area. In addition, the light detector 300b removes noise of light emitted from the second aspherical lens 330b between the second aspherical lens 330b and the optical analyzer 350b, and the second aspherical surface. The device may further include a photodiode integrated circuit (PDIC) 340b disposed to amplify the light emitted from the lens 330b. By using the photodiode integrated device 340b, it is possible to further reduce the size of the PCR device and to measure a reliable optical signal by minimizing noise.
도 4i에 따르면, 상기 PCR 장치는 상기 광 제공부(200b)로부터 방출된 광이 광 검출부(300b)까지 도달할 수 있도록 광의 진행 방향을 조절하고, 미리 결정된 파장을 갖는 광을 분리하기 위한 하나 이상의 이색성 필터(400x, 400y)를 더 포함한다. 상기 이색성 필터(dichroic filter)(400x, 400y)는 광을 파장에 따라 선택적으로 투과 또는 선택적으로 조절된 각도로 반사시키는 모듈이다. 도 4k에 따르면, 이색성 필터(400x)는 광 제공부(200b)로부터 방출되는 광의 광축에 대하여 약 45도 각도로 경사지게 배치되고, 상기 광을 그 파장에 따라 선택적으로 단파장 성분을 투과시키고 장파장 성분을 직각으로 반사시켜 상기 광 투과성 열 블록(100b) 상에 배치된 PCR 칩(10)에 도달하게 한다. 또한, 이색성 필터(400y)는 상기 PCR 칩(10) 및 광 투과성 열 블록(100b)으로부터 반사된 광의 광축에 대하여 약 45도 각도로 경사지게 배치되고, 상기 광을 그 파장에 따라 선택적으로 단파장 성분을 투과시키고 장파장 성분을 직각으로 반사시켜 상기 광 검출부(300b)에 도달하게 한다. 상기 광 검출부(300b)에 도달한 광은 광 분석기에서 전기 신호로 전환되어 핵산 증폭 여부 및 증폭 정도를 나타내게 된다.According to FIG. 4I, the PCR apparatus may adjust one or more directions of light so that light emitted from the light providing unit 200b may reach the light detecting unit 300b and separate one or more light having a predetermined wavelength. It further includes dichroic filters 400x and 400y. The dichroic filters 400x and 400y are modules that reflect light at an angle selectively transmitted or selectively adjusted according to the wavelength. According to FIG. 4K, the dichroic filter 400x is disposed to be inclined at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light providing unit 200b, and selectively transmits the short wavelength component according to its wavelength and transmits the long wavelength component. Is reflected at right angles to reach the PCR chip 10 disposed on the light transmitting thermal block 100b. In addition, the dichroic filter 400y is disposed to be inclined at an angle of about 45 degrees with respect to the optical axis of the light reflected from the PCR chip 10 and the light transmitting thermal block 100b, and the light is selectively shortened according to its wavelength. And the long wavelength component is reflected at right angles to reach the light detector 300b. The light reaching the light detector 300b is converted into an electrical signal in the optical analyzer to indicate whether the nucleic acid is amplified and the degree of amplification.
도 5a 내지 도 5d는 본 발명의 제6 실시예에 따른 PCR 장치에 관한 도면이다.5A to 5D are diagrams of a PCR device according to a sixth embodiment of the present invention.
도 5a 내지 도 5d에 따른 본 발명의 제6 실시예에 따른 PCR 장치는 1 이상의 히터를 구비하는 히터 군, 상기 히터 군을 2 이상 구비하고 상기 2 이상의 히터 군은 상호 열 교환이 일어나지 않도록 이격 배치된 히터 유닛이 2 이상 반복 배치된 것으로서, 적어도 일 면에 표적 샘플이 수용되는 PCR 칩의 접촉 면을 구비하는 열 블록; 상기 열 블록에 구비된 히터들에 전력을 공급하도록 연결된 전극을 구비하는 전극부; 및 상기 열 블록에 구비된 1 이상의 히터들과 열 교환이 가능하도록 상기 열 블록 상에 접촉가능하도록 배치된 본 발명의 제3 실시예에 따른 PCR 칩(10)을 포함한다.PCR apparatus according to a sixth embodiment of the present invention according to Figures 5a to 5d is provided with a heater group having at least one heater, at least two heater groups and the two or more heater groups are spaced apart so that mutual heat exchange does not occur Two or more heater units, the thermal block including a contact surface of a PCR chip to accommodate a target sample on at least one surface; An electrode unit having an electrode connected to supply electric power to heaters provided in the thermal block; And a PCR chip 10 according to a third embodiment of the present invention disposed to be in contact with the heat block to allow heat exchange with one or more heaters provided in the heat block.
상기 열 블록(100c)은 PCR을 수행하기 위해 표적 샘플에 특정 온도로 열을 공급하도록 구현된 모듈으로서, 적어도 일 면에 표적 샘플이 수용되는 PCR 칩(10)의 접촉 면을 구비하고, 상기 PCR 칩(10)의 일 면에 접촉하여, 1 이상의 반응 채널 내에 존재하는 표적 샘플에 열을 공급하여 PCR을 수행하도록 한다. 상기 열 블록(100c)은 기판을 기반으로 한다. 상기 기판은 상기 기판 내에 배치된 히터의 가열 및 온도 유지로 인해 그 물리적 및/또는 화학적 성질이 변하지 않고, 상기 기판 내에 이격 배치된 2 이상의 히터 사이에서 상호 열 교환이 일어나지 않도록 하는 모든 재질로 구현될 수 있다. 예를 들어, 상기 기판은 플라스틱, 유리, 실리콘 등의 재질로서, 투명 또는 반투명하게 구현될 수 있으나, 본 발명의 제5 실시예에 따른 PCR 장치가 실시간(real-time) PCR 용도로 사용될 경우 광 투과성 재질로 구현되는 것이 바람직하다. 상기 열 블록(100c)은 전체적으로 평면 형상을 구비할 수 있으나, 이에 제한되는 것은 아니다. 상기 열 블록(100c)은 1 이상의 히터를 구비하는 히터 군, 상기 히터 군을 2 이상 구비하고 상기 2 이상의 히터 군은 상호 열 교환이 일어나지 않도록 이격 배치된 히터 유닛이 2 이상 반복 배치된다. 또한, 상기 PCR 칩(10)의 접촉 면은 상기 열 블록(100c)의 적어도 일 면에 구현되고, 표적 샘플이 수용된 PCR 칩(10)에 효율적으로 열을 공급하기 위한 다양한 형상으로 구현될 수 있으나, 접촉 면의 표면적이 넓도록 평면 형상 또는 필러(pillar) 형상이 바람직하다. 상기 히터(111c, 112c, 121c, 122c, 131c, 132c)는 발열 소자로서, 그 내부에 열선(도시되지 않음)이 배치될 수 있다. 상기 열선은 일정 온도를 유지하도록 다양한 열원과 구동가능하게 연결될 수 있고, 상기 열선의 온도를 모니터링하기 위한 다양한 온도 센서와 구동가능하게 연결될 수 있다. 상기 열선은 상기 히터의 내부 온도를 전체적으로 일정하게 유지하기 위해 상기 히터의 표면 중심점을 기준으로 상하 및/또는 좌우 방향으로 대칭되도록 배치될 수 있다. 또한, 상기 히터는 그 내부에 박막 히터(thin film heater, 도시되지 않음)가 배치될 수도 있다. 상기 박막 히터는 상기 히터의 내부 온도를 전체적으로 일정하게 유지하기 위해 상기 히터 표면의 중심점을 기준으로 상하 및/또는 좌우 방향으로 일정한 간격으로 배치될 수 있다. 또한, 상기 히터는 발열 소자로서, 동일한 면적에 대한 고른 열 분포 및 신속한 열 전달을 위한 그 자체로 금속 재질, 예를 들어 크롬, 알루미늄, 구리, 철, 은 등일 수 있다. 또한, 상기 히터는 광 투과성 발열 소자, 예를 들어 산화물 반도체 물질 또는 상기 산화물 반도체 물질에 In, Sb, Al, Ga, C 및 Sn로 구성된 군으로부터 선택된 불순물이 첨가된 물질을 포함하는 도전성 나노 입자, 인듐 주석 산화물, 전도성 고분자 물질, 탄소 나노 튜브, 및 그래핀(graphene)이 포함된 군으로부터 선택되는 하나 이상을 포함할 수 있다. 만약 상기 PCR 장치가 실시간(real-time) PCR 용도로 사용될 경우 상기 히터는 광 투과성 발열 소자인 것이 바람직하다. 상기 히터 군(110c, 120c, 130c)은 상기 1 이상의 히터를 포함하는 단위로서, PCR 수행을 위한 변성 단계, 어닐링 단계 및/또는 연장 단계를 수행하기 위한 온도를 유지하는 영역이다. 상기 히터 군은 상기 열 블록(100c)에 2 이상 배치되고, 상기 2 이상의 히터 군은 상호 열 교환이 일어나지 않도록 이격 배치된다. 상기 히터 군은 상기 열 블록(100c)에 2개 내지 4개 포함될 수 있다. 즉, 상기 열 블록은 2개의 히터 군을 구비하고, 상기 제1 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제2 히터 군은 PCR 어닐링/연장 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 어닐링/연장 단계 온도를 유지하고 상기 제2 히터 군은 PCR 변성 단계 온도를 유지할 수 있다. 또한, 상기 열 블록은 3개의 히터 군을 구비하고, 상기 제1 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제2 히터 군은 PCR 어닐링 단계 온도를 유지하고 상기 제3 히터 군은 PCR 연장 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 어닐링 단계 온도를 유지하고 상기 제2 히터 군은 PCR 연장 단계 온도를 유지하고 상기 제3 히터 군은 PCR 변성 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 연장 단계 온도를 유지하고 상기 제2 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제3 히터 군은 PCR 어닐링 단계 온도를 유지할 수 있다. 바람직하게는 상기 히터 군은 상기 열 블록(100c)에 3회 배치되어 PCR 수행을 위한 3 단계, 즉 변성 단계, 어닐링 단계 및 연장 단계를 수행하기 위한 온도를 각각 유지할 수 있고, 더 바람직하게는 상기 히터 군은 상기 열 블록(100c)에 2회 배치되어 PCR 수행을 위한 2 단계, 즉 변성 단계 및 어닐링/연장 단계를 수행하기 위한 온도를 각각 유지할 수 있으나, 이에 제한되는 것은 아니다. 상기 히터 군은 상기 열 블록(100c)에 2회 배치되어 PCR 수행을 위한 2 단계, 즉 변성 단계 및 어닐링/연장 단계를 수행할 경우 PCR 수행을 위한 3 단계, 즉 변성 단계, 어닐링 단계 및 연장 단계를 수행하는 것보다 반응 시간을 단축할 수 있고, 히터의 수를 줄임으로써 구조를 단순화시키는 이점이 있다. 이 경우 PCR 수행을 위한 3 단계에 있어서, 변성 단계를 수행하기 위한 온도는 85℃ 내지 105℃, 바람직하게는 95℃이고, 어닐링 단계를 수행하기 위한 온도는 40℃ 내지 60℃, 바람직하게는 50℃이고, 연장 단계를 수행하기 위한 온도는 50℃ 내지 80℃, 바람직하게는 72℃이고, PCR 수행을 위한 2 단계에 있어서, 변성 단계를 수행하기 위한 온도는 85℃ 내지 105℃, 바람직하게는 95℃이고, 어닐링/연장 단계를 수행하기 위한 온도는 50℃ 내지 80℃, 바람직하게는 72℃이다. 다만, 상기 PCR 수행을 위한 특정된 온도 및 온도 범위는 PCR을 수행함에 있어서 실현 가능한 범위 내에서 조절 가능하다. 한편, 상기 히터 군은 온도 완충 역할을 수행하는 히터를 더 포함할 수 있다. 상기 히터 유닛(10c, 20c)은 상기 1 이상의 히터를 포함하는 상기 2 이상의 히터 군을 포함하는 단위로서, PCR 수행을 위한 변성 단계, 어닐링 단계 및/또는 연장 단계를 포함하는 1 순환이 완료되는 영역이다. 상기 히터 유닛은 상기 열 블록(100c)에 2 이상 반복 배치된다. 바람직하게는 상기 히터 유닛은 상기 열 블록(100c)에 10회, 20회, 30회 또는 40회로 반복 배치될 수 있으나, 이에 제한되는 것은 아니다.The thermal block 100c is a module implemented to supply heat to a target sample at a specific temperature to perform a PCR. The thermal block 100c includes a contact surface of a PCR chip 10 on which at least one surface a target sample is accommodated. In contact with one side of the chip 10, PCR is performed by supplying heat to a target sample present in one or more reaction channels. The thermal block 100c is based on a substrate. The substrate may be made of any material such that physical and / or chemical properties thereof do not change due to heating and temperature maintenance of a heater disposed in the substrate, and mutual heat exchange does not occur between two or more heaters spaced apart in the substrate. Can be. For example, the substrate may be made of plastic, glass, silicon, or the like, and may be implemented transparently or semitransparently. However, when the PCR device according to the fifth embodiment of the present invention is used for real-time PCR, It is preferred to be implemented with a transparent material. The thermal block 100c may have a planar shape as a whole, but is not limited thereto. The heat block 100c includes a heater group including one or more heaters, two or more heater groups, and the two or more heater groups are repeatedly arranged at least two heater units spaced apart from each other so that mutual heat exchange does not occur. In addition, the contact surface of the PCR chip 10 may be implemented on at least one surface of the thermal block 100c and may be implemented in various shapes for efficiently supplying heat to the PCR chip 10 in which a target sample is accommodated. For example, a planar shape or a pillar shape is preferable so that the surface area of a contact surface is wide. The heaters 111c, 112c, 121c, 122c, 131c, and 132c are heat generating elements, and heat wires (not shown) may be disposed therein. The heating wire may be operably connected with various heat sources to maintain a constant temperature, and may be operably connected with various temperature sensors for monitoring the temperature of the heating wire. The heating wire may be arranged to be symmetrical in the vertical direction and / or the horizontal direction with respect to the surface center point of the heater in order to maintain the internal temperature of the heater as a whole. Also, the heater may have a thin film heater (not shown) disposed therein. The thin film heater may be disposed at regular intervals in the vertical direction and / or the left and right directions with respect to the center point of the heater surface in order to maintain the internal temperature of the heater as a whole. In addition, the heater is a heating element, and may itself be a metal material, for example, chromium, aluminum, copper, iron, silver, and the like, for even heat distribution and rapid heat transfer over the same area. In addition, the heater is a light-transmitting heating element, for example, conductive nanoparticles including an oxide semiconductor material or a material added with impurities selected from the group consisting of In, Sb, Al, Ga, C and Sn to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive polymeric materials, carbon nanotubes, and graphene. If the PCR device is used for real-time PCR, the heater is preferably a light transmitting heating element. The heater groups 110c, 120c, and 130c are units including one or more heaters, and are regions that maintain a temperature for performing a denaturation step, annealing step, and / or an extension step for PCR. Two or more heater groups are disposed in the heat block 100c, and the two or more heater groups are spaced apart from each other so that mutual heat exchange does not occur. Two to four heater groups may be included in the thermal block 100c. That is, the thermal block includes two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature. In addition, the thermal block includes three heater groups, wherein the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group has the PCR extension step temperature. Or the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater The group may maintain the PCR extension step temperature, the second heater group may maintain the PCR denaturation step temperature, and the third heater group may maintain the PCR annealing step temperature. Preferably, the heater group may be disposed three times in the thermal block 100c to maintain three temperatures for performing PCR, that is, a temperature for performing a denaturation step, an annealing step, and an extension step, and more preferably, The heater group may be disposed twice in the thermal block 100c to maintain two temperatures for performing PCR, that is, denaturation and annealing / extension, respectively, but are not limited thereto. The heater group is disposed in the heat block 100c twice, and when performing the two steps for performing PCR, that is, the denaturation step and the annealing / extension step, three steps for performing the PCR, i. It is possible to shorten the reaction time rather than to perform, there is an advantage of simplifying the structure by reducing the number of heaters. In this case, in the three steps for performing the PCR, the temperature for performing the denaturation step is 85 ℃ to 105 ℃, preferably 95 ℃, the temperature for performing the annealing step is 40 ℃ to 60 ℃, preferably 50 ℃, the temperature for performing the extension step is 50 ℃ to 80 ℃, preferably 72 ℃, in two steps for performing the PCR, the temperature for performing the denaturation step is 85 ℃ to 105 ℃, preferably 95 ° C., and the temperature for performing the annealing / extension step is 50 ° C. to 80 ° C., preferably 72 ° C. However, the specified temperature and temperature range for performing the PCR can be adjusted within a range feasible in performing the PCR. On the other hand, the heater group may further include a heater that serves as a temperature buffer. The heater units 10c and 20c are units including the two or more heater groups including the one or more heaters, and an area in which one cycle including a denaturation step, annealing step, and / or extension step for performing PCR is completed. to be. The heater unit is repeatedly arranged at least two in the heat block 100c. Preferably, the heater unit may be repeatedly arranged in the heat block 100c 10 times, 20 times, 30 times, or 40 times, but is not limited thereto.
도 5a에 따르면, 상기 열 블록(100c)은 반복 배치된 히터 유닛(10c, 20c), 그에 각각 포함된 2개의 히터 군(110c, 120c), 및 그에 각각 포함된 1개의 히터(111c, 121c)를 구비함으로써, PCR 수행을 위한 2 단계 온도, 즉 변성 단계의 1 온도 및 어닐링/연장 단계의 1 온도를 순차적으로 반복 제공한다. 예를 들어, 제1 히터(111c)는 85℃ 내지 105℃ 범위 중 1 온도, 바람직하게는 95℃를 유지하여 상기 제1 히터 군(110c)은 변성 단계를 수행하기 위한 온도를 제공하고, 제2 히터(121c)는 50℃ 내지 80℃ 범위 중 1 온도, 바람직하게는 72℃를 유지하여 상기 제2 히터 군(120c)은 어닐링/연장 단계를 수행하기 위한 온도를 제공함으로써, 상기 열 블록(100c)은 제1 히터 유닛(10c) 및 제2 히터 유닛(20c)에서 PCR 수행을 위한 2 단계 온도를 순차적으로 반복 제공한다.According to FIG. 5A, the heat block 100c includes heater units 10c and 20c that are repeatedly arranged, two heater groups 110c and 120c included therein, and one heater 111c and 121c respectively included therein. By providing a two-step temperature for performing the PCR, that is, one temperature of the denaturation step and one temperature of the annealing / extension step are sequentially provided. For example, the first heater 111c maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C. such that the first heater group 110c provides a temperature for performing the modification step. The second heater 121c maintains one temperature in the range of 50 ° C. to 80 ° C., preferably 72 ° C. such that the second heater group 120 c provides a temperature for performing the annealing / extension step. 100c) sequentially and repeatedly provides a two-step temperature for performing PCR in the first heater unit 10c and the second heater unit 20c.
도 5b에 따르면, 상기 열 블록(100c)은 반복 배치된 히터 유닛(10c, 20c), 그에 각각 포함된 2개의 히터 군(110c, 120c), 및 그에 각각 포함된 2개의 히터(111c, 112c, 121c, 122c)를 구비함으로써, PCR 수행을 위한 2 단계 온도, 즉 변성 단계의 2 온도 및 어닐링/연장 단계의 2 온도를 순차적으로 반복 제공한다. 예를 들어, 제1 히터(111c)는 85℃ 내지 105℃ 범위 중 1 온도, 제2 히터(112c)는 85℃ 내지 105℃ 범위 중 상기 제1 히터(111c)의 온도와 동일한 또는 상이한 1 온도를 유지하여 상기 제1 히터 군(110c)은 변성 단계를 수행하기 위한 온도를 제공하고, 제3 히터(121c)는 50℃ 내지 80℃ 범위 중 1 온도, 제4 히터(122c)는 50℃ 내지 80℃ 범위 중 상기 제3 히터(121c)의 온도와 동일한 또는 상이한 1 온도를 유지하여 상기 제2 히터 군(120c)은 어닐링/연장 단계를 수행하기 위한 온도를 제공함으로써, 상기 열 블록(100c)은 제1 히터 유닛(10c) 및 제2 히터 유닛(20c)에서 PCR 수행을 위한 2 단계 온도를 순차적으로 반복 제공한다.According to FIG. 5B, the heat block 100c includes heater units 10c and 20c that are repeatedly arranged, two heater groups 110c and 120c included therein, and two heaters 111c and 112c respectively included therein. 121c, 122c) provides two steps of temperature for performing PCR, that is, two temperatures of the denaturation step and two temperatures of the annealing / extension step. For example, the first heater 111c has one temperature in the range of 85 ° C to 105 ° C, and the second heater 112c has one temperature that is the same as or different from the temperature of the first heater 111c in the range of 85 ° C to 105 ° C. By maintaining the first heater group 110c provides a temperature for performing the modification step, the third heater 121c is one temperature in the range of 50 ℃ to 80 ℃, the fourth heater 122c is 50 ℃ to The thermal block 100c may be maintained by maintaining a temperature equal to or different from the temperature of the third heater 121c in the range of 80 ° C. such that the second heater group 120c provides a temperature for performing an annealing / extension step. Provides sequentially two-step temperature for performing PCR in the first heater unit 10c and the second heater unit 20c.
도 5c에 따르면, 상기 열 블록(100c)은 반복 배치된 히터 유닛(10c, 20c), 그에 각각 포함된 3개의 히터 군(110c, 120c, 130c), 및 그에 각각 포함된 1개의 히터(111c, 121c, 131c)를 구비함으로써, PCR 수행을 위한 3 단계 온도, 즉 변성 단계의 1 온도, 어닐링 단계의 1 온도, 및 연장 단계의 1 온도를 순차적으로 반복 제공한다. 예를 들어, 제1 히터(111c)는 85℃ 내지 105℃ 범위 중 1 온도, 바람직하게는 95℃를 유지하여 상기 제1 히터 군(110c)은 변성 단계를 수행하기 위한 온도를 제공하고, 제2 히터(121c)는 40℃ 내지 60℃ 범위 중 1 온도, 바람직하게는 50℃를 유지하여 상기 제2 히터 군(120c)은 어닐링 단계를 수행하기 위한 온도를 제공하고, 제3 히터(131c)는 50℃ 내지 80℃ 범위 중 1 온도, 바람직하게는 72℃를 유지하여 상기 제3 히터 군(130c)은 연장 단계를 수행하기 위한 온도를 제공함으로써, 상기 열 블록(100c)은 제1 히터 유닛(10c) 및 제2 히터 유닛(20c)에서 PCR 수행을 위한 3 단계 온도를 순차적으로 반복 제공한다.According to FIG. 5C, the heat block 100c includes heater units 10c and 20c that are repeatedly arranged, three heater groups 110c, 120c and 130c respectively included therein, and one heater 111c respectively included therein. 121c and 131c) sequentially provide three step temperatures for performing PCR, that is, one temperature of the denaturation step, one temperature of the annealing step, and one temperature of the extension step. For example, the first heater 111c maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C. such that the first heater group 110c provides a temperature for performing the modification step. The second heater 121c maintains one temperature, preferably 50 ° C, in the range of 40 ° C to 60 ° C so that the second heater group 120c provides a temperature for performing the annealing step, and the third heater 131c Is maintained at a temperature in the range of 50 ° C to 80 ° C, preferably 72 ° C, so that the third heater group 130c provides a temperature for performing the extension step, whereby the thermal block 100c is configured as a first heater unit. In step 10c and the second heater unit 20c, three steps of temperature for performing PCR are repeatedly provided sequentially.
도 5d에 따르면, 반복 배치된 히터 유닛(10c, 20c), 그에 각각 포함된 3개의 히터 군(110c, 120c, 130c), 및 그에 각각 포함된 2개의 히터(111c, 112c, 121c, 122c, 131c, 132c)를 구비함으로써, PCR 수행을 위한 3 단계 온도, 즉 변성 단계의 2 온도, 어닐링 단계의 2 온도, 및 연장 단계의 2 온도를 순차적으로 반복 제공한다. 예를 들어, 제1 히터(111c)는 85℃ 내지 105℃ 범위 중 1 온도, 제2 히터(112c)는 85℃ 내지 105℃ 범위 중 상기 제1 히터(111c)의 온도와 동일한 또는 상이한 1 온도를 유지하여 상기 제1 히터 군(110c)은 변성 단계를 수행하기 위한 온도를 제공하고, 제3 히터(121c)는 40℃ 내지 60℃ 범위 중 1 온도, 제4 히터(122c)는 40℃ 내지 60℃ 범위 중 상기 제3 히터(121c)의 온도와 동일한 또는 상이한 1 온도를 유지하여 제2 히터 군(120c)은 어닐링 단계를 수행하기 위한 온도를 제공하고, 제5 히터(131c)는 50℃ 내지 80℃ 범위 중 1 온도, 제6 히터(132c)는 50℃ 내지 80℃ 범위 중 상기 제5 히터(131c)의 온도와 동일한 또는 상이한 1 온도를 유지하여 상기 제3 히터 군(130c)은 연장 단계를 수행하기 위한 온도를 제공함으로써, 상기 열 블록(100c)은 제1 히터 유닛(10c) 및 제2 히터 유닛(20c)에서 PCR 수행을 위한 3 단계 온도를 순차적으로 반복 제공한다.According to FIG. 5D, heater units 10c and 20c that are repeatedly arranged, three heater groups 110c, 120c and 130c included therein, and two heaters 111c, 112c, 121c, 122c and 131c respectively included therein are included. , 132c), provides three steps of temperature for performing PCR, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step. For example, the first heater 111c has one temperature in the range of 85 ° C to 105 ° C, and the second heater 112c has one temperature that is the same as or different from the temperature of the first heater 111c in the range of 85 ° C to 105 ° C. By maintaining the first heater group 110c provides a temperature for performing the modification step, the third heater 121c is one temperature in the range of 40 ℃ to 60 ℃, the fourth heater 122c is 40 ℃ to The second heater group 120c provides a temperature for performing the annealing step by maintaining one temperature equal to or different from the temperature of the third heater 121c in the range of 60 ° C, and the fifth heater 131c is 50 ° C. The sixth heater 132c in the range of 1 to 80 ° C. maintains one temperature that is the same or different from the temperature of the fifth heater 131c in the range of 50 ° C. to 80 ° C. so that the third heater group 130c extends. By providing a temperature for performing the step, the thermal block 100c performs PCR in the first heater unit 10c and the second heater unit 20c. A third step for temperature provides successively repeated.
도 5a 내지 5d와 같이, 일정 온도를 유지하는 2 이상의 히터를 반복 배치함으로써 온도 변화율을 크게 개선할 수 있다. 예를 들어, 종래 하나의 히터만을 채택하는 단일 히터 방식에 의하면, 온도 변화율이 초당 3℃ 내지 7℃ 범위 내에서 이루어지는데 반해, 본 발명의 제6 실시예에 따른 반복 히터 배치 방식에 의하면, 상기 히터들 간의 온도 변화율이 초당 20℃ 내지 40℃ 범위 내에서 이루어져 반응 시간을 크게 단축할 수 있다. 상기 히터들은 상호 열 교환이 일어나지 않도록 이격 배치되어 있고, 그 결과, 미세한 온도 변화에 의해서도 큰 영향을 받을 수 있는 핵산 증폭 반응에 있어서, 상기 변성 단계, 어닐링 단계 및 연장 단계(또는 상기 변성 단계 및 어닐링/변성 단계)의 정확한 온도 제어가 가능하고, 상기 히터들로부터 열을 공급받는 부위에서만 원하는 온도 또는 온도 범위를 유지하는 것이 가능하다. 또한, 상기 열 블록(100c)에는 상기 히터 유닛이 2 이상 반복 배치되어 있고, 상기 히터 유닛(10c, 20c)의 반복 배치 수는 PCR을 수행하고자 하는 사용자 또는 표적 샘플의 종류에 따라 다양할 수 있다. 예를 들어, 본 발명의 제5 실시예에 따른 PCR 장치를 순환 주기 10회로 하는 PCR에 적용하고자 하는 경우 상기 히터 유닛을 10회 반복 배치할 수 있다. 즉, PCR을 수행하고자 하는 사용자 또는 표적 샘플의 종류에 따라 PCR 순환 주기를 고려하여 상기 히터 유닛을 10회, 20회, 30회, 40회, 50회 등으로 반복 배치할 수 있고, 이는 특별히 제한되는 것은 아니다. 한편, 상기 히터 유닛을 미리 결정된 PCR 순환 주기의 절반의 수로 반복 배치할 수도 있다. 예를 들어, 본 발명의 일 실시예에 따른 PCR 장치를 순환 주기 20회로 하는 PCR에 적용하고자 하는 경우 상기 히터 유닛을 10회 반복 배치할 수 있다. 이 경우 표적 샘플 용액은 이하 상세하게 설명될 핵산 증폭 반응부(300c) 내에 배치된 반응 채널(303c) 내에서 유입부(304c)로부터 유출부(305c) 방향으로 PCR 순환 주기를 10회 반복 실행하되, 뒤이어 반대로 유출부(305c)로부터 유입부(304c) 방향으로 PCR 순환 주기를 10회 반복 실행할 수 있다.5A to 5D, the rate of change of temperature can be greatly improved by repeatedly disposing two or more heaters maintaining a constant temperature. For example, according to the conventional single heater method using only one heater, the temperature change rate is within the range of 3 ° C to 7 ° C per second, whereas according to the repeating heater arrangement method according to the sixth embodiment of the present invention, The rate of change of temperature between the heaters is within the range of 20 ℃ to 40 ℃ per second can greatly shorten the reaction time. The heaters are spaced apart so that mutual heat exchange does not occur, and as a result, in the nucleic acid amplification reaction that can be greatly affected by minute temperature changes, the denaturation step, annealing step and extension step (or the denaturation step and annealing). Accurate temperature control), and it is possible to maintain the desired temperature or temperature range only at the site where heat is supplied from the heaters. In addition, two or more heater units are repeatedly arranged in the thermal block 100c, and the number of repetitive arrangements of the heater units 10c and 20c may vary according to the type of a user or a target sample to perform PCR. . For example, when the PCR apparatus according to the fifth embodiment of the present invention is to be applied to a PCR having ten cycles, the heater unit may be repeatedly arranged ten times. That is, the heater unit may be repeatedly arranged in 10 times, 20 times, 30 times, 40 times, 50 times, etc. in consideration of the PCR circulation cycle according to the type of the user or target sample to be PCR, which is particularly limited. It doesn't happen. On the other hand, the heater unit may be repeatedly arranged in half of the predetermined PCR cycle. For example, when a PCR device according to an embodiment of the present invention is to be applied to a PCR having a circulation cycle of 20 cycles, the heater unit may be repeatedly arranged ten times. In this case, the target sample solution is repeated 10 times the PCR cycle from the inlet 304c to the outlet 305c in the reaction channel 303c disposed in the nucleic acid amplification reaction unit 300c to be described in detail below. On the contrary, the PCR cycle may be repeated 10 times from the outlet portion 305c toward the inlet portion 304c.
상기 전극부는 전력 공급부(도시되지 않음)로부터 전력을 공급받아 상기 열 블록(100c)이 발열되도록 전력을 공급하는 모듈로서, 상기 열 블록(100c)에 구비된 히터들에 전력을 공급하도록 연결된 전극을 포함할 수 있다. 상기 열 블록(100c)의 제1 전극은 상기 제1 히터(110c)에 전력을 공급하도록 연결되고, 상기 제2 전극은 상기 제2 히터(120c)에 전력을 공급하도록 연결되어 있으나, 이에 제한되지 않고 상기 전극부는 상기 열 블록(100c)의 외부에 구동가능하게 배치될 수도 있다. 만약 상기 제1 히터(110c)가 PCR 변성 단계 온도, 예를 들어 85℃ 내지 105℃를 유지하고 상기 제2 히터(120c)가 PCR 어닐링/연장 단계 온도, 예를 들어 50℃ 내지 80℃를 유지하는 경우 상기 제1 전극은 전력 공급부로부터 PCR 변성 단계 온도 유지를 위한 전력을 공급받고, 상기 제2 전극은 전력 공급부로부터 PCR 어닐링/연장 단계 온도 유지를 위한 전력을 공급받을 수 있다. 상기 제1 전극 및 상기 제2 전극은 상기 열 블록(100c)에 반복 배치된 제1 히터(110c) 및 2 이상의 제2 히터(120c)에 각각 연결될 수 있다. 상기 제1 전극 및 상기 제2 전극은 금, 은, 구리 등 전도성 재질일 수 있고, 특별히 제한되는 것은 아니다.The electrode unit is a module that receives power from a power supply unit (not shown) and supplies power to heat the heat block 100c. The electrode is connected to supply an electric power to the heaters provided in the heat block 100c. It may include. The first electrode of the thermal block 100c is connected to supply power to the first heater 110c, and the second electrode is connected to supply power to the second heater 120c, but is not limited thereto. Instead, the electrode unit may be disposed to be driven outside the thermal block 100c. If the first heater 110c maintains the PCR denaturation step temperature, for example 85 ℃ to 105 ℃ and the second heater 120c maintains the PCR annealing / extension stage temperature, for example 50 ℃ to 80 ℃ In this case, the first electrode may receive power for maintaining the PCR denaturation step temperature from the power supply, and the second electrode may receive power for maintaining the PCR annealing / extension step temperature from the power supply. The first electrode and the second electrode may be connected to the first heater 110c and the two or more second heaters 120c that are repeatedly disposed in the thermal block 100c. The first electrode and the second electrode may be a conductive material such as gold, silver, copper, and the like, and are not particularly limited.
상기 PCR 칩(10)은 상기 열 블록(100c)과 열 교환이 가능하도록 상기 열 블록(100c) 상에 접촉하여 상기 제1 히터(110c) 및 상기 제2 히터(120c)로부터 열을 공급받을 수 있다. 또한, 상기 PCR 칩(10)은 상기 제1 히터(110c)의 상측 대응 부분(301c) 및 상기 제2 히터(120c)의 상측 대응 부분(302c)을 길이 방향으로 통과하도록 연장 배치된 1 이상의 반응 채널(14)을 구비할 수 있다. 상기 반응 채널(14)은 상기 열 블록(100c)에 배치된 상기 제1 히터(110c)의 상측 대응 부분과 상기 제2 히터(120c)의 상측 대응 부분이 유체 소통가능하게 연결되어 길이 방향으로 통과하도록 연장되어 있다. 상기 반응 채널(14)의 상기 제1 히터(110c)의 상측 대응 부분은 표적 샘플 내에 존재하는 핵산의 PCR 변성 반응이 일어나는 영역이고, 상기 제2 히터(120c)의 상측 대응 부분은 표적 샘플 내에 존재하는 핵산의 PCR 어닐링/연장 반응이 일어나는 영역일 수 있다. 즉, 상기 반응 채널(14)을 유동하는 표적 샘플이 상기 제1 히터(110c)의 상측 대응 부분 및 상기 제2 히터(120c)의 상측 대응 부분을 연이어 통과하면서 PCR이 수행될 수 있다. 또한, 상기 PCR 칩(10)은 상기 1 이상의 반응 채널(14)의 양 말단에 각각 유입부(15) 및 유출부(16)를 포함할 수 있다. 한편, 상기 1 이상의 반응 채널(14)은 상기 히터 유닛 중 최선 배치된 제1 히터(110c)의 상측 대응 부분과 최후 배치된 제2 히터(120c)의 상측 대응 부분을 직선 길이 방향으로 통과하도록 연장 배치되는 것이 바람직하다. 따라서 상기 유입부(15)를 통해 유입된 표적 샘플은 상기 반응 채널(14)을 길이 방향으로 통과하면서 상기 제1 히터(110c)의 상측 대응 부분 및 상기 제2 히터의 상측 대응 부분에서 각각 PCR 변성 단계 및 PCR 어닐링/연장 단계를 반복 수행하며 상기 유출부를 통해 외부로 배출될 수 있다. 한편, 상기 PCR 칩(10)은 전체적으로 평면 형상일 수 있으나, 이에 제한되는 것은 아니다. 한편, 상기 PCR 칩(10)은 광 투과성 재질로 구현될 수 있고, 만약 본 발명의 제5 실시예에 따른 PCR 장치가 실시간(real-time) PCR 용도로 사용될 경우 상기 PCR 칩(10)은 광 투과성 재질로 구현되는 것이 바람직하다. The PCR chip 10 may be supplied with heat from the first heater 110c and the second heater 120c in contact with the heat block 100c to allow heat exchange with the heat block 100c. have. In addition, the PCR chip 10 is one or more reactions extending to extend in the longitudinal direction through the upper corresponding portion 301c of the first heater (110c) and the upper corresponding portion (302c) of the second heater (120c) Channel 14 may be provided. The reaction channel 14 passes in the longitudinal direction by connecting the upper corresponding portion of the first heater 110c disposed in the thermal block 100c and the upper corresponding portion of the second heater 120c in fluid communication. Is extended. The upper corresponding portion of the first heater 110c of the reaction channel 14 is a region where PCR denaturation reaction of nucleic acid present in the target sample occurs, and the upper corresponding portion of the second heater 120c is present in the target sample. It may be a region where PCR annealing / extension reaction of the nucleic acid occurs. That is, PCR may be performed while the target sample flowing through the reaction channel 14 passes through the upper corresponding portion of the first heater 110c and the upper corresponding portion of the second heater 120c in succession. In addition, the PCR chip 10 may include an inlet 15 and an outlet 16 at both ends of the one or more reaction channels 14, respectively. On the other hand, the one or more reaction channels 14 extend so as to pass through the upper corresponding portion of the first heater 110c disposed best of the heater unit and the upper corresponding portion of the second heater 120c disposed last in a straight length direction. It is preferable to arrange. Therefore, the target sample introduced through the inlet part 15 passes through the reaction channel 14 in the longitudinal direction, and is respectively modified with PCR in the upper corresponding part of the first heater 110c and the upper corresponding part of the second heater. Repeating the step and PCR annealing / extension step may be discharged to the outside through the outlet. On the other hand, the PCR chip 10 may be a planar shape as a whole, but is not limited thereto. On the other hand, the PCR chip 10 may be implemented with a light transmissive material, if the PCR device according to the fifth embodiment of the present invention is used for real-time (real-time PCR) the PCR chip 10 is optical It is preferred to be implemented with a transparent material.
실험예 1. 혈청 타입별 구제역 바이러스 플라스미드 DNA 합성 및 제작Experimental Example 1. Synthesis and Preparation of Foot-and-mouth Virus Plasmid DNA by Serum Type
Genbank로부터 구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)에 관한 유전정보를 획득하고, 각 혈청 타입별 염기서열을 코스모진텍㈜에 의뢰하여 유전자 합성을 진행하고, Vector pUC57 내에 합성한 유전자를 삽입하여 클로닝(cloning)하였다. 또한, sequencing하여 삽입된 유전자의 정확도를 확인하였다. DH5α(박테리아)를 사용하여 형질전환(Transformation)을 하여 배양 플레이트에 도말 후 올바르게 클로닝(cloning)된 콜로니(Colony)만 선별 및 재배양하여 플라스미드(plasmid) DNA를 추출하였다. 추출된 플라스미드(plasmid) DNA를 시퀀싱(Sequencing)하여 클로닝(cloning)한 플라스미드(plasmid) DNA의 염기서열을 분석하였고 유전자 서열에 맞게 증폭된 것을 확인하였다.Foot and mouth virus A (Genbank ID number: NC011450), foot and mouth virus O (Genbank ID number: NC004004), foot and mouth virus C (Genbank ID number: NC002554), foot and mouth virus Asia (Genbank ID number: NC004915), foot and mouth virus SAT 1 from Genbank (Genbank ID number: NC011451), and the genetic information on the foot-and-mouth virus SAT 2 (Genbank ID number: NC011452) was obtained, and the base sequence of each serotype was commissioned by Cosmogenetech Co., Ltd. to proceed with gene synthesis, Vector pUC57 The synthesized gene was inserted into and cloned. In addition, the accuracy of the inserted gene was confirmed by sequencing. Transformation was performed using DH5α (bacteria) to plasmid DNA by selecting and culturing only colonies that were cloned correctly after plating on culture plates. The sequencing of the extracted plasmid DNA was performed to analyze the nucleotide sequence of the plasmid DNA cloned (cloning) and confirmed that it was amplified according to the gene sequence.
실험예 2. 구제역 바이러스 검출용 프라이머 세트 제작 및 합성Experimental Example 2. Preparation and synthesis of primer set for foot-and-mouth virus detection
Genbank로부터 획득한 혈청 타입별 염기서열을 기초로 CLC main workbench version 6.6(Denmark)을 이용하여 sequence alignment를 수행하고, 이를 기초로 GC(%)를 50 내지 65%가 되도록 하고, Tm(℃) 값 70 내지 72℃ 조건이 되도록 하여 Primer 3를 통해 본 발명의 일 실시예에 따른 프라이머 세트를 제작하였다. 제작된 프라이머 세트는 아래 표 1과 같다.Sequence alignment is performed using CLC main workbench version 6.6 (Denmark) based on the serotype base sequence obtained from Genbank, and based on this, GC (%) is 50 to 65%, Tm (℃) value To prepare a primer set according to an embodiment of the present invention through Primer 3 to 70 to 72 ℃ conditions. The prepared primer set is shown in Table 1 below.
표 1
번호 염기서열(5'→3') Tm(℃) GC(%) Product size(bp)
서열번호 1 CAC GCC GTG GGA CYA THC AGG A 72.7 63.6 92
서열번호 3 CAC GCC GTG GGA CCA TAC AGG A
서열번호 2 GGG YTC RAA GAG RCG CCG GTA Y 71.5 63.6
서열번호 4 GGG CTC AAA GAG ACG CCG GTA C
Table 1
number Sequence (5 '→ 3') Tm (℃) GC (%) Product size (bp)
SEQ ID NO: 1 CAC GCC GTG GGA CYA THC AGG A 72.7 63.6 92
SEQ ID NO: 3 CAC GCC GTG GGA CCA TAC AGG A
SEQ ID NO: 2 GGG YTC RAA GAG RCG CCG GTA Y 71.5 63.6
SEQ ID NO: 4 GGG CTC AAA GAG ACG CCG GTA C
실험예 3. 본 발명의 제4 구체예에 따른 PCR 장치 및 타사의 PCR 장치를 이용한 구제역 바이러스의 실시간 검출Experimental Example 3. Real-time detection of foot-and-mouth virus using a PCR device according to a fourth embodiment of the present invention and other companies' PCR devices
혈청 타입별 구제역 원인 바이러스에 대한 프라이머 세트의 동시적이고 특이적인 검출을 확인하기 위해, 실시예 1에서 합성된 플라스미드 DNA를 주형으로 하고, 아래 표 2 및 표 3의 조건에 따라 PCR 샘플 용액을 제작하였다. 표 2의 PCR 샘플 용액 조건은 타사의 PCR 장치(Bio-Rad:CFX-connect, 이하 같다)에 사용하기 위한 것이고, 표 3의 PCR 샘플 용액 조건은 본 발명의 제4 실시예에 따른 PCR 장치에 사용하기 위한 것이다.In order to confirm simultaneous and specific detection of primer sets for foot-and-mouth disease-causing viruses by serum type, the plasmid DNA synthesized in Example 1 was used as a template, and PCR sample solutions were prepared according to the conditions of Tables 2 and 3 below. . PCR sample solution conditions of Table 2 are for use in other companies' PCR device (Bio-Rad: CFX-connect, below), and PCR sample solution conditions of Table 3 is applied to the PCR device according to the fourth embodiment of the present invention. It is to use.
표 2
조성 부피(㎕)
TOYOBO SYBR qPCR mix 10
Forward Primer(10μM) 2
Reverse Primer(10μM) 2
Template DNA 1
증류수(DW) 5
Total 20
TABLE 2
Furtherance Volume (μl)
TOYOBO SYBR qPCR mix 10
Forward Primer (10 μM) 2
Reverse Primer (10 μM) 2
Template DNA One
Distilled Water (DW) 5
Total 20
표 3
조성 부피(㎕)
NBS 2X master mix 8
Forward Primer(10μM) 1.6
Reverse Primer(10μM) 1.6
Template DNA 1
증류수(DW) 3.8
Total 16
TABLE 3
Furtherance Volume (μl)
NBS 2X master mix 8
Forward Primer (10 μM) 1.6
Reverse Primer (10 μM) 1.6
Template DNA One
Distilled Water (DW) 3.8
Total 16
뒤이어, 아래 표 4 및 표 5의 PCR 조건에 따라 본 발명의 제4 실시예에 따른 PCR 장치 및 타사의 PCR 장치를 이용하여 각각 PCR을 수행하였다. 표 4의 PCR 조건은 타사의 PCR 장치에 적용한 것이고, 표 5의 PCR 조건은 본 발명의 제4 실시예에 따른 PCR 장치에 적용한 것이다.Subsequently, PCR was performed using the PCR apparatus according to the fourth embodiment of the present invention and the third-party PCR apparatus according to the PCR conditions of Tables 4 and 5 below. The PCR conditions of Table 4 are applied to the PCR device of the other company, the PCR conditions of Table 5 is applied to the PCR device according to the fourth embodiment of the present invention.
표 4
온도(℃) 시간(sec) 사이클(cycle)
Pre-denaturation 95℃ 1800(30 min) 1
Denaturation 95℃ 5 40
Annealing & Extension 72-65℃ 30
Table 4
Temperature (℃) Time (sec) Cycle
Pre-denaturation 95 ℃ 1800 (30 min) One
Denaturation 95 ℃ 5 40
Annealing & Extension 72-65 ℃ 30
표 5
온도(℃) 시간(sec) 사이클(cycle)
Pre-denaturation 95℃ 8 1
Denaturation 95℃ 3 40
Annealing & Extension 72-64℃ 6
Table 5
Temperature (℃) Time (sec) Cycle
Pre-denaturation 95 ℃ 8 One
Denaturation 95 ℃ 3 40
Annealing & Extension 72-64 ℃ 6
실험 결과Experiment result
PCR 비교실험 결과는 도 6 내지 11에 의해 상세하게 확인할 수 있다. 구체적으로, 도 6은 구제역 바이러스 A 플라스미드 시료를 대상으로 하는 PCR 비교실험 결과이고, 도 7은 구제역 바이러스 O 플라스미드 시료를 대상으로 하는 PCR 비교실험 결과이고, 도 8은 구제역 바이러스 C 플라스미드 시료를 대상으로 하는 PCR 비교실험 결과이고, 도 9는 구제역 바이러스 Asia 플라스미드 시료를 대상으로 하는 PCR 비교실험 결과이고, 도 10은 구제역 바이러스 SAT 1 플라스미드 시료를 대상으로 하는 PCR 비교실험 결과이며, 도 11은 구제역 바이러스 SAT 2 플라스미드 시료를 대상으로 하는 PCR 비교실험 결과이다. 또한, 도 6 내지 11에 있어서, 도 a 내지 b는 각각 타사의 PCR 장치 및 본 발명의 제4 실시예에 따른 PCR 장치를 이용한, 음성 대조군 대비 혈청 타입별 구제역 바이러스 플라스미드 시료 농도별 Ct 값에 관한 표이고, 도 c 내지 d는 각각 타사의 PCR 장치 및 본 발명의 제4 실시예에 따른 PCR 장치에 의한 PCR 증폭 산물의 사이클별(cycles, x축) 형광도(Fluorescence, y축) 변화에 관한 그래프이며, 도 e 내지 f는 각각 타사의 PCR 장치 및 본 발명의 제4 실시예에 따른 PCR 장치에 의한 PCR 증폭 산물에 관한 전기영동 사진이다. 위 비교실험에 따르면, 상당한 시간이 소요된 타사의 PCR 장치의 PCR 결과와 상대적으로 훨씬 빨리 반응을 종료한 본 발명의 제4 실시예에 따른 PCR 장치의 PCR 결과가 큰 차이가 없거나 또는 상당히 신뢰도가 높다는 것을 확인할 수 있다(Ct 값 비교).The results of PCR comparison experiments can be confirmed in detail by FIGS. 6 to 11. Specifically, FIG. 6 is a result of PCR comparison with the foot-and-mouth virus A plasmid sample, FIG. 7 is a result of PCR comparison with the foot-and-mouth virus O plasmid sample, and FIG. 8 shows a foot-and-mouth virus C plasmid sample. 9 is a result of PCR comparison experiment for the foot-and-mouth virus virus plasmid sample, FIG. 10 is a result of PCR comparison for the foot-and-mouth virus SAT 1 plasmid sample, and FIG. 11 is a foot-and-mouth virus SAT It is the result of PCR comparison experiment with 2 plasmid samples. In addition, in FIGS. 6 to 11, FIGS. A to b each show a Ct value of each foot-and-mouth disease virus plasmid sample concentration compared to a negative control group using a third-party PCR device and a PCR device according to a fourth embodiment of the present invention. Tables c to d show changes in cycles (x-axis) fluorescence (y-axis) of PCR amplification products by a third-party PCR device and a PCR device according to a fourth embodiment of the present invention, respectively. Figures e to f are electrophoresis pictures of the PCR amplification products by the third-party PCR device and the PCR device according to the fourth embodiment of the present invention, respectively. According to the above comparative experiments, the PCR results of the PCR device according to the fourth embodiment of the present invention, which completed the reaction relatively much faster than the PCR results of the other company's PCR device, which took a considerable time, were not significantly different or were highly reliable. It can be confirmed that it is high (compare Ct values).

Claims (24)

  1. 서열번호 1의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머 및 서열번호 2의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머로 이루어진,Consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2,
    구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)로 구성된 군으로부터 1 이상 선택된 유전자를 검출하기 위한,Foot-and-mouth disease virus A (Genbank ID number: NC011450), foot-and-mouth virus O (Genbank ID number: NC004004), foot-and-mouth virus C (Genbank ID number: NC002554), foot-and-mouth virus Asia (Genbank ID number: NC004915), foot-and-mouth virus SAT 1 (Genbank ID number: NC011451), and to detect one or more selected genes from the group consisting of foot and mouth virus SAT 2 (Genbank ID number: NC011452),
    혈청 타입별 구제역(Foot and mouth disease, FMD) 검출용 프라이머 세트.Primer set for detecting foot and mouth disease (FMD) by serum type.
  2. 서열번호 3의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머 및 서열번호 4의 염기 서열 중 15개 이상의 연속적인 뉴클레오티드를 포함하는 프라이머로 이루어진,Consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 4,
    구제역 바이러스 A(Genbank ID number: NC011450), 구제역 바이러스 O(Genbank ID number: NC004004), 구제역 바이러스 C(Genbank ID number: NC002554), 구제역 바이러스 Asia(Genbank ID number: NC004915), 구제역 바이러스 SAT 1(Genbank ID number: NC011451), 및 구제역 바이러스 SAT 2(Genbank ID number: NC011452)로 구성된 군으로부터 1 이상 선택된 유전자를 검출하기 위한,Foot-and-mouth disease virus A (Genbank ID number: NC011450), foot-and-mouth virus O (Genbank ID number: NC004004), foot-and-mouth virus C (Genbank ID number: NC002554), foot-and-mouth virus Asia (Genbank ID number: NC004915), foot-and-mouth virus SAT 1 (Genbank ID number: NC011451), and to detect one or more selected genes from the group consisting of foot and mouth virus SAT 2 (Genbank ID number: NC011452),
    혈청 타입별 구제역(Foot and mouth disease, FMD) 검출용 프라이머 세트.Primer set for detecting foot and mouth disease (FMD) by serum type.
  3. 제1 판; First edition;
    상기 제1 판 상에 배치되고, 1 이상의 반응 채널을 구비하는 제2 판; 및 A second plate disposed on the first plate and having one or more reaction channels; And
    상기 제2 판 상에 배치되고, 상기 1 이상의 반응 채널의 양 말단과 연결되되 개폐 가능하도록 구현된 유입부 및 유출부를 구비하는 제3 판을 포함하는 것으로서,A third plate disposed on the second plate, the third plate having an inlet and an outlet connected to both ends of the one or more reaction channels and configured to be openable and closed;
    상기 1 이상의 반응 채널 내에 제1항 또는 제2항에 따른 프라이머 세트를 포함하는 것을 특징으로 하는 PCR(Polymerase Chain Reaction) 칩.Polymer chain reaction (PCR) chip comprising a primer set according to claim 1 or 2 in the at least one reaction channel.
  4. 제3항에 있어서,The method of claim 3,
    상기 제1 판 및 제3 판은 폴리디메틸실옥산(polydimethylsiloxane, PDMS), 사이클로올레핀코폴리머(cycle olefin copolymer, COC), 폴리메틸메타크릴레이트(polymethylmetharcylate, PMMA), 폴리카보네이트(polycarbonate, PC), 폴리프로필렌카보네이트(polypropylene carbonate, PPC), 폴리에테르설폰(polyether sulfone, PES), 및 폴리에틸렌텔레프탈레이트(polyethylene terephthalate, PET), 및 그의 조합물로 구성된 군으로부터 선택되는 재질을 포함하고, The first and third plates are polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (PMMA), polycarbonate (PC), A material selected from the group consisting of polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof,
    상기 제2 판은 폴리메틸메타크릴레이트(polymethylmethacrylate, PMMA), 폴리카보네이트(polycarbonate, PC), 사이클로올레핀 코폴리머(cycloolefin copolymer, COC), 폴리아미드(polyamide, PA), 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리페닐렌 에테르(polyphenylene ether, PPE), 폴리스티렌(polystyrene, PS), 폴리옥시메틸렌(polyoxymethylene, POM), 폴리에테르에테르케톤(polyetheretherketone, PEEK), 폴리테트라프로오르에틸렌(polytetrafluoroethylene, PTFE), 폴리비닐클로라이드(polyvinylchloride, PVC), 폴리비닐리덴 플로라이드(polyvinylidene fluoride, PVDF), 폴리부틸렌테레프탈레이트(polybutyleneterephthalate, PBT), 불소화에틸렌프로필렌(fluorinated ethylenepropylene, FEP), 퍼플로로알콕시알칸(perfluoralkoxyalkane, PFA), 및 그의 조합물로 구성된 군으로부터 선택되는 열 가소성 수지 또는 열 경화성 수지 재질을 포함하는 것을 특징으로 하는 PCR 칩.The second plate is polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (CCO), polyamide (PA), polyethylene (PE, PE), Polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetheretherketone (PEEK), polytetraproethylene (polytetrafluoroethylene, PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT), fluorinated ethylenepropylene (FEP), perflow Thermoplastic or thermosetting resin material selected from the group consisting of perfluoralkoxyalkane (PFA), and combinations thereof PCR chip, characterized in that.
  5. 제3항에 있어서,The method of claim 3,
    상기 PCR 칩은 플라스틱 재질로 구현되되, 광 투과성을 갖도록 구현된 것을 특징으로 하는 PCR 칩.The PCR chip is implemented as a plastic material, PCR chip, characterized in that implemented to have a light transmission.
  6. 제3항에 있어서, The method of claim 3,
    상기 1 이상의 반응 채널 내에 dATP, dCTP, dGTP, 및 dTTP를 포함하는 혼합물, DNA 중합효소 및 검출 가능한 표지를 더 포함하는 것을 특징으로 하는 PCR 칩.The PCR chip further comprises a mixture comprising dATP, dCTP, dGTP, and dTTP, DNA polymerase and detectable label in the one or more reaction channels.
  7. 제6항에 있어서,The method of claim 6,
    상기 검출 가능한 표지는 Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 및 티아졸 오렌지(thiazole orange)로 이루어진 군으로부터 선택되는 것을 특징으로 하는 PCR 칩.The detectable label is Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2 , Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43 , SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX PCR chip, characterized in that selected from the group consisting of Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 and thiazole orange.
  8. 기판 상에 배치된 제1 열 블록;A first thermal block disposed on the substrate;
    상기 기판 상에 상기 제1 열 블록과 이격 배치된 제2 열 블록; 및A second thermal block spaced apart from the first thermal block on the substrate; And
    상기 제1 열 블록 및 제2 열 블록 위로 구동 수단에 의해 좌우 및/또는 상하 이동 가능하고, 제3항 내지 제7항 중 어느 한 항에 따른 PCR 칩이 장착된 칩 홀더;A chip holder movable left and right and / or up and down by a driving means over the first row block and the second row block, and on which the PCR chip according to any one of claims 3 to 7 is mounted;
    를 포함하는 PCR 장치.PCR device comprising a.
  9. 제8항에 있어서, The method of claim 8,
    상기 제1 열 블록과 제2 열 블록 사이에 광원이 더 배치되고, 상기 칩 홀더 위에 상기 광원으로부터 방출되는 광을 검출하기 위한 광 검출부가 더 배치되거나, 또는 상기 제1 열 블록과 제2 열 블록 사이에 광원으로부터 방출되는 광을 검출하기 위한 광 검출부가 더 배치되고, 상기 칩 홀더 위에 광원이 더 배치되는 것을 특징으로 하는 PCR 장치.A light source is further disposed between the first row block and the second row block, and a light detector for detecting light emitted from the light source is further disposed on the chip holder, or the first row block and the second row block. And a light detector for detecting light emitted from the light source in between, and a light source further disposed on the chip holder.
  10. 기판, 상기 기판 상에 배치된 도전성 나노 입자를 포함하는 발열층, 상기 발열층 상에 배치된 절연 보호층 및 상기 발열층과 연결 배치된 전극을 구비하되, 광투과성을 갖도록 구현된 광투과성 열 블록; 및A light-transmitting heat block comprising a substrate, a heat generating layer including conductive nanoparticles disposed on the substrate, an insulating protective layer disposed on the heat generating layer, and an electrode disposed in connection with the heat generating layer, wherein the heat transmitting layer is formed to have light transparency. ; And
    상기 광투과성 열 블록의 상부 면에 접촉 가능하도록 배치된, 제3항 내지 제7항 중 어느 한 항에 따른 PCR 칩;A PCR chip according to any one of claims 3 to 7, arranged to be in contact with an upper surface of the light transmitting thermal block;
    을 포함하는 PCR 장치.PCR device comprising a.
  11. 제10항에 있어서,The method of claim 10,
    상기 기판은 광 투과성 유리 또는 플라스틱 재질이고, 상기 발열층에 포함된 도전성 나노 입자는 산화물 반도체 물질 또는 상기 산화물 반도체 물질에 In, Sb, Al, Ga, C 및 Sn로 구성된 군으로부터 선택된 불순물이 첨가된 물질이고, 상기 절연 보호층은 유전체 산화물, 페릴린, 나노 입자 및 고분자 필름으로 구성된 군으로부터 선택되는 것이고, 상기 전극은 금속 물질, 전도성 에폭시, 전도성 페이스트, 솔더 및 전도성 필름으로 구성된 군으로부터 선택되는 것을 특징으로 하는 PCR 장치.The substrate is a light-transmissive glass or plastic material, the conductive nanoparticles included in the heating layer is an oxide semiconductor material or an impurity selected from the group consisting of In, Sb, Al, Ga, C and Sn is added to the oxide semiconductor material Material, and the insulating protective layer is selected from the group consisting of dielectric oxide, perylene, nanoparticles, and polymer film, and the electrode is selected from the group consisting of metal material, conductive epoxy, conductive paste, solder, and conductive film. PCR device characterized in that.
  12. 제10항에 있어서, The method of claim 10,
    상기 광투과성 열 블록의 기판의 하부 면은 흡광 물질이 포함된 흡광층이 접촉 배치되거나, 또는 상기 광투과성 열 블록의 절연 보호층의 상부 면은 광반사 방지 물질이 포함된 광반사방지층이 접촉 배치된 것을 특징으로 하는 PCR 장치.The lower surface of the substrate of the light-transmissive thermal block is disposed in contact with the light absorbing layer containing the light absorbing material, or the upper surface of the insulating protective layer of the light-transmissive thermal block is in contact with the light reflection preventing layer containing light-reflective material PCR device characterized in that.
  13. 제10항에 있어서, The method of claim 10,
    상기 PCR 장치는 상기 칩 접촉부에 배치되는 PCR 칩에 광을 제공하도록 구동가능하게 배치된 광 제공부 및 상기 칩 접촉부에 배치되는 PCR 칩으로부터 방출되는 광을 수용하도록 구동가능하게 배치된 광 검출부를 더 포함하는 것을 특징으로 하는 PCR 장치.The PCR device further includes a light providing unit operably arranged to provide light to the PCR chip disposed in the chip contact unit and a light detection unit operatively arranged to receive light emitted from the PCR chip disposed in the chip contact unit. PCR device comprising a.
  14. 1 이상의 히터를 구비하는 히터 군, 상기 히터 군을 2 이상 구비하고 상기 2 이상의 히터 군은 상호 열 교환이 일어나지 않도록 이격 배치된 히터 유닛이 2 이상 반복 배치된 것으로서, 적어도 일 면에 표적 샘플이 수용되는 PCR 칩의 접촉 면을 구비하는 열 블록;Two or more heater groups including at least one heater group and at least two heater groups, wherein at least one heater unit is spaced apart from each other so that mutual heat exchange does not occur. A thermal block having a contact surface of the PCR chip;
    상기 열 블록에 구비된 히터들에 전력을 공급하도록 연결된 전극을 구비하는 전극부; 및An electrode unit having an electrode connected to supply electric power to heaters provided in the thermal block; And
    상기 열 블록에 구비된 1 이상의 히터들과 열 교환이 가능하도록 상기 열 블록 상에 접촉가능하도록 배치된, 제3항 내지 제7항 중 어느 한 항에 따른 PCR 칩;The PCR chip according to any one of claims 3 to 7, arranged to be in contact with the heat block to enable heat exchange with one or more heaters provided in the heat block;
    을 포함하는 PCR 장치.PCR device comprising a.
  15. 제14항에 있어서, 상기 열 블록은 2개 내지 4개의 히터 군을 구비하는 것을 특징으로 하는 PCR 장치.15. The PCR device according to claim 14, wherein the thermal block has two to four heater groups.
  16. 제14항에 있어서,The method of claim 14,
    상기 열 블록은 2개의 히터 군을 구비하고, 상기 제1 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제2 히터 군은 PCR 어닐링/연장 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 어닐링/연장 단계 온도를 유지하고 상기 제2 히터 군은 PCR 변성 단계 온도를 유지하는 것을 특징으로 하는 PCR 장치.The thermal block has two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group is PCR annealing And / or maintain the extension step temperature and the second heater group maintains the PCR denaturation step temperature.
  17. 제14항에 있어서,The method of claim 14,
    상기 열 블록은 3개의 히터 군을 구비하고, 상기 제1 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제2 히터 군은 PCR 어닐링 단계 온도를 유지하고 상기 제3 히터 군은 PCR 연장 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 어닐링 단계 온도를 유지하고 상기 제2 히터 군은 PCR 연장 단계 온도를 유지하고 상기 제3 히터 군은 PCR 변성 단계 온도를 유지하거나, 또는 상기 제1 히터 군은 PCR 연장 단계 온도를 유지하고 상기 제2 히터 군은 PCR 변성 단계 온도를 유지하고 상기 제3 히터 군은 PCR 어닐링 단계 온도를 유지하는 것을 특징으로 하는 PCR 장치.The thermal block has three heater groups, the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group maintains the PCR extension step temperature. Alternatively, the first heater group maintains the PCR annealing step temperature, the second heater group maintains the PCR extension step temperature, and the third heater group maintains the PCR denaturation step temperature, or the first heater group PCR apparatus, characterized in that it maintains the PCR extension step temperature, the second heater group maintains the PCR denaturation step temperature and the third heater group maintains the PCR annealing step temperature.
  18. 제14항에 있어서,The method of claim 14,
    상기 열 블록은 광 투과성을 갖도록 구현된 것을 특징을 하는 PCR 장치.PCR device, characterized in that the thermal block is implemented to have a light transmittance.
  19. 제14항에 있어서,The method of claim 14,
    상기 열 블록에 구비된 히터는 광 투과성 발열소자를 포함하는 것을 특징으로 하는 PCR 장치.The PCR device, characterized in that the heater provided in the thermal block comprises a light transmitting heat generating element.
  20. 제14항에 있어서,The method of claim 14,
    상기 전극부에 전력을 공급하기 위한 전력 공급부 및 상기 1 이상의 반응 채널 내에서 유동하는 유체의 유량 및 유속을 제어하기 위해 양압 또는 음압을 제공하도록 배치된 펌프를 더 포함하는 것을 특징으로 하는 PCR 장치.And a pump arranged to provide a positive pressure or a negative pressure to control a flow rate and a flow rate of the fluid flowing in the at least one reaction channel, and a power supply for supplying power to the electrode part.
  21. 제14항에 있어서, The method of claim 14,
    상기 제1 히터 및 상기 제2 히터 사이에 광원이 배치되고, 상기 전극부에 전력을 공급하기 위한 전력 공급부, 상기 1 이상의 반응 채널 내에서 유동하는 유체의 유량 및 유속을 제어하기 위해 양압 또는 음압을 제공하도록 배치된 펌프, 및 상기 광원으로부터 방출되는 광을 검출하기 위한 광 검출부를 더 포함하는 것을 특징으로 하는 PCR 장치.A light source is disposed between the first heater and the second heater, a power supply unit for supplying power to the electrode unit, positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the one or more reaction channels And a pump arranged to provide, and a light detector for detecting light emitted from the light source.
  22. 제14항에 있어서, The method of claim 14,
    상기 전극부에 전력을 공급하기 위한 전력 공급부, 상기 1 이상의 반응 채널 내에서 유동하는 유체의 유량 및 유속을 제어하기 위해 양압 또는 음압을 제공하도록 배치된 펌프, 상기 PCR 칩에 광을 제공하도록 배치된 광 제공부, 및 상기 PCR 칩으로부터 방출되는 광을 수용하도록 배치된 광 검출부를 더 포함하는 것을 특징으로 하는 PCR 장치.A power supply for supplying power to the electrode portion, a pump arranged to provide a positive pressure or a negative pressure to control the flow rate and flow rate of the fluid flowing in the one or more reaction channels, the pump arranged to provide light to the PCR chip And a light providing unit, and a light detecting unit arranged to receive light emitted from the PCR chip.
  23. 구제역 감염이 의심되는 대상 시료를 제3항 내지 제7항 중 어느 한 항에 따른 PCR 칩의 상기 1 이상의 반응 채널에 도입하여 PCR을 수행하는 단계; 및Performing a PCR by introducing a target sample suspected of foot and mouth infection into the one or more reaction channels of the PCR chip according to any one of claims 3 to 7; And
    상기 PCR 결과로부터 상기 대상 시료 중에 구제역 바이러스의 존재 유무를 확인하는 단계;Confirming the presence or absence of foot-and-mouth virus in the target sample from the PCR result;
    를 포함하는 구제역 검출 방법.Foot-and-mouth disease detection method comprising a.
  24. 제23항에 있어서,The method of claim 23, wherein
    상기 PCR 수행 단계는 제8항 내지 제9항 중 어느 한 항에 따른 PCR 장치, 제10항 내지 제13항 중 어느 한 항에 따른 PCR 장치, 및 제14항 내지 제22항 중 어느 한 항에 따른 PCR 장치로 구성된 군으로부터 선택된 PCR 장치에서 수행되는 것을 특징으로 하는 구제역 검출 방법.The PCR step is carried out in any one of the PCR device according to any one of claims 8 to 9, the PCR device according to any one of claims 10 to 13, and any one of claims 14 to 22. Foot-and-mouth disease detection method characterized in that performed in a PCR device selected from the group consisting of a PCR device according to.
PCT/KR2013/004189 2012-05-30 2013-05-10 Primer set for detecting foot and mouth disease according to serum type, pcr device using same, and method for detecting foot and mouth disease by using same WO2013180406A1 (en)

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