WO2014104770A1 - Ensemble d'amorces pour la détection d'une intoxication alimentaire, appareil de pcr à l'aide de celui-ci, et procédé de détection d'une intoxication alimentaire à l'aide de ceux-ci - Google Patents

Ensemble d'amorces pour la détection d'une intoxication alimentaire, appareil de pcr à l'aide de celui-ci, et procédé de détection d'une intoxication alimentaire à l'aide de ceux-ci Download PDF

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WO2014104770A1
WO2014104770A1 PCT/KR2013/012237 KR2013012237W WO2014104770A1 WO 2014104770 A1 WO2014104770 A1 WO 2014104770A1 KR 2013012237 W KR2013012237 W KR 2013012237W WO 2014104770 A1 WO2014104770 A1 WO 2014104770A1
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pcr
light
syto
heater
chip
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PCT/KR2013/012237
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English (en)
Korean (ko)
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김성우
이세현
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나노바이오시스(주)
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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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a primer set for detecting food poisoning bacteria, a PCR chip comprising the same, a PCR device including the same, and a method for simultaneously detecting various food poisoning bacteria using the same.
  • a method for quickly and economically checking the presence of food poisoning bacteria in a sample such as food is required.
  • a common method for detecting food poisoning bacteria is to cultivate a sample in a selective medium, isolate bacteria suspected of food poisoning bacteria, and then confirm this by biochemical or immunological methods.
  • immunological methods using antibodies can detect bacteria with high accuracy, a large amount of samples are required, and protein purification, production, or peptide production of the bacteria is essential for producing antibodies required for each diagnosis. High antibody production costs are required.
  • recently used real-time PCR method is a method for observing the increase of the PCR amplification product in real time every cycle of the PCR, a method for interpreting the detection and quantification of the fluorescent material reacted with the PCR amplification product.
  • This method eliminates the need for additional electrophoresis, excellent accuracy, high sensitivity, high reproducibility, and automation, compared to conventional PCR methods, which have been stained on gels after final steps to identify PCR amplification products after electrophoresis. It is possible to quantify the results, to be quick and easy, to be excellent in biological safety due to contamination by dyes such as EtBr (Ethidium Bromide) and harmful problems such as UV irradiation, and to automatically check whether a specific gene is amplified.
  • EtBr Ethidium Bromide
  • One embodiment of the present invention is to provide a primer set for detecting food poisoning bacteria that can detect at least one or more of the five food poisoning bacteria.
  • a PCR chip for detecting food poisoning bacteria comprising a primer set for detecting food poisoning bacteria that can detect at least one or more of the five food poisoning bacteria in the PCR chip and a PCR device comprising the same.
  • another embodiment of the present invention is to provide a method for detecting several types of food poisoning bacteria in real time using a PCR chip for detecting food poisoning bacteria and a PCR device including the same.
  • a first embodiment of the present invention is Enteropathogenic E. coli consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 1 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: Primer sets for detecting the eaeA gene of EPEC); Detecting the invA gene of Enteroinvasive E.
  • EIEC EIEC coli
  • a primer set for detecting ceuE gene of Campylobacter coli consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 6
  • a primer set for detecting the ompW gene of Vibrio cholerae consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 8
  • a primer set for detecting the Vvh gene of Vibrio vulnificus consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of S
  • a second embodiment of the present invention comprises a 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 and 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 (cycle olefin copolymer (COC), polymethyl methacrylate (polymethylmetharcylate, PMMA) ), Polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof
  • the second plate is 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), polybuty
  • 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 third 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 which is movable left, right, and / or up and down by a driving means over the first row block and the second row block, and is equipped with a PCR chip according to the second 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 fourth embodiment of the present invention includes a substrate, a heating layer including conductive nanoparticles disposed on the substrate, an insulating protective layer disposed on the heating layer, and an electrode disposed in connection with the heating layer, wherein the light transmitting A light transmissive thermal block implemented to have; And it provides a PCR device comprising a PCR chip according to a second 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 such 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 second 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 thermal 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 sixth embodiment of the present invention includes the steps of performing a PCR by introducing a target sample suspected of food poisoning infection into the one or more reaction channels of the PCR chip according to the second embodiment of the present invention; And it provides a food poisoning bacteria detection method comprising the step of confirming the presence or absence of food poisoning bacteria 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 PCR chip comprising the same, a PCR device including the same, and a food poisoning bacteria detection method using the same, it is inexpensive to detect food poisoning bacteria infection. Accurate and rapid identification at cost can greatly contribute to preventing and spreading food poisoning bacteria.
  • FIG. 1 is a diagram of a PCR chip according to a second embodiment of the present invention.
  • FIG. 2 is a diagram of a PCR chip according to a second 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 third embodiment of the present invention.
  • FIGS. 4A to 4I are diagrams of a PCR device according to a fourth embodiment of the present invention.
  • 5A to 5H are diagrams of a PCR device according to a fifth embodiment of the present invention.
  • 6a to 6b are primer sets of SEQ ID NOs: 1 and 2, primer sets of SEQ ID NOs: 3 and 4, primer sets of SEQ ID NOs: 5 and 6, primer sets of SEQ ID NOs: 7 and 8, primer sets of SEQ ID NOs: 9 and 10
  • electrophoresis photographs showing real-time PCR results of a sample using a PCR device of another company and a PCR device according to a third embodiment of the present invention to be.
  • 7A and 7B show primer sets of SEQ ID NOs: 1 and 2, primer sets of SEQ ID NOs: 3 and 4, primer sets of SEQ ID NOs: 5 and 6, primer sets of SEQ ID NOs: 7 and 8, primer sets of SEQ ID NOs: 9 and 10 It is a graph showing a real-time PCR results for a sample using a PCR device of another company and a PCR device according to a third embodiment of the present invention, assuming a primer set for detecting food poisoning bacteria according to an embodiment of the present invention .
  • Primer for detecting food poisoning bacteria is 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
  • EIEC EIEC coli
  • a primer set for detecting ceuE gene of Campylobacter coli consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 6
  • a primer set for detecting the ompW gene of Vibrio cholerae consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 8
  • a primer set for detecting the Vvh gene of Vibrio vulnificus consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of S
  • 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 primers are typically 15 to 30 nucleotides, although varying 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 set according to the first embodiment of the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence that is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the sequence and acting as a primer. .
  • 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 primers are the same as SEQ ID NO: 1 and SEQ ID NO: 2 (SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10).
  • the primer is the SEQ ID NO: 1 and SEQ ID NO: 2 (SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10) may be an oligonucleotide having a nucleotide sequence of any one.
  • the size of the PCR product of the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 according to the first embodiment of the present invention was designed to 238 bp (base pair), the size of the PCR product of the primer set of SEQ ID NO: 3 and SEQ ID NO: 4 was designed at 216 bp (base pair), the PCR product of SEQ ID NO: 5 and SEQ ID NO: 6 primer set was designed to 234 bp (base pair), PCR product of SEQ ID NO: 7 and SEQ ID NO: 8
  • the size of was designed to 249 bp (base pair)
  • the size of the PCR product of the primer set of SEQ ID NO: 9 and SEQ ID NO: 10 was designed to 213 bp (base pair).
  • the method for detecting food poisoning bacteria using the primer set is as follows. First, PCR is performed by introducing a target sample suspected of food poisoning infection into the one or more reaction channels of a PCR chip which will be described in detail below.
  • Subject sample means a sample (sample or sample solution) of an individual expected to be infected with food poisoning bacteria, and includes, but is not limited to, for example, cultured cells, blood, saliva, and the like.
  • the target sample suspected of food poisoning infection may include extracting DNA from the sample. Thereafter, the extracted DNA is introduced into a PCR chip to perform real-time PCR. Therefore, the presence or absence of food poisoning bacteria 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. 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.
  • a PCR chip may include 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 15 and an outlet 16 connected to both ends of the one or more reaction channels 14 and configured to be opened and closed. 13), comprising in said at least one reaction channel 14 a primer set according to a first embodiment of the invention.
  • deoxyribonucleotide triphosphates dNTP
  • dNTP deoxyribonucleotide triphosphates
  • 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 planar 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 the 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 plastic thickness, 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 upper surface of the first plate 11 is treated with a hydrophilic material 17 can perform the 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 range of use of the PCR device, according to Figure 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 of the unit.
  • the third plate 13 may be formed 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 portion 16 may have various sizes, but preferably may be selected from 1.0 mm to 1.5 mm in diameter.
  • the inlet 15 and the outlet 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. You can prevent it.
  • the cover means may be implemented in various shapes, sizes or materials.
  • the thickness of the third plate may vary, but preferably may 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 in the plate material having a size corresponding to the lower surface of the third plate 13 to the outlet portion 16 of the third plate 13 Mechanically forming one or more reaction channels 14 to a corresponding portion to provide a second plate 12; Providing a first plate (11) by forming a surface made of 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., to single-strand the double stranded DNA.
  • 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 the primers of the partial DNA-primer complex by DNA polymerase for example at 72 ° C.
  • the DNA having the specific base sequence can be exponentially amplified.
  • 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, the PCR device refers to an apparatus including modules for performing the above steps, and detailed modules not described herein are all provided in the scope of the related art disclosed and obvious 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 second embodiment of the present invention may be 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 symmetrically 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 thermal block 100a and 200a in order to maintain a constant internal temperature of the first and second thermal 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 thermal block 100a and the second thermal 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. The denaturation occurs to reduce the efficiency of the PCR efficiency or the reaction may not occur, if the denaturation step temperature is higher than 100 °C the enzyme used in the PCR loses the activity, 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, and 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, the denaturation step and the nucleic acid amplification reaction may be significantly affected by a slight temperature change. 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 up and down movement of the driving means 500a may be controlled by control means (not shown) disposed operably 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 the annealing and extension (or amplification) steps. 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 third 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, an 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
  • an 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) steps 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 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 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.
  • the light detector 800a may be disposed on the driving means 500a, and the through means 530a may be disposed on 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 in the PCR chip 10 can be detected in real time during the nucleic acid amplification reaction by the PCR device 1.
  • a separate fluorescent substance may be further added to the sample solution introduced into the PCR chip 10.
  • the light source 700a is disposed to be as wide as possible in the spaced space between the first column block 100a and the second column block 200a and is arranged to emit the same light as much as possible.
  • the light source 700a may be operably connected to a lens (not shown) that collects light emitted from the light source 700a and an optical filter (not shown) that filters 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 performed by 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 pass through the driving means 500a, specifically, the through part 530a disposed on the rail 510a to reach the light exit part 800a. 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
  • FIGS. 4A to 4I are diagrams illustrating a PCR device according to a fourth embodiment of the present invention.
  • FIG. 4A illustrates a light transmissive thermal block 100b included in a PCR device according to a fourth 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 second 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 insulating protective 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 portion 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 apparatus including the light transmitting thermal block 100b has many advantages over the conventional PCR apparatus 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 fourth 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 transmissive thermal block 100b through the electrode 40b to observe a temperature change of the light transmissive 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 fourth 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 shows a light transmissive thermal block 100b included in a PCR device according to a fourth embodiment of the invention in which a light reflection prevention layer 70b is disposed in contact with an upper surface thereof
  • FIG. 4e shows a lower 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 placed 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 fourth embodiment of the present invention is shown.
  • PCR 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.
  • PCR is called real time PCR.
  • 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 heat 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 to provide the light to the PCR chip 10. Module for measuring the PCR reaction performed.
  • the PCR device According to the PCR device according to the fourth embodiment of the present invention, the amplification of nucleic acid (phosphorescent substance 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 or not the amplification of the target nucleic acid contained in the initial sample solution.
  • 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 an 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. 230b), and a first optical lens 240b for collecting light emitted from the first light filter, and disposed 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.
  • the first light filter 230b may pass only light having a wavelength 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 portion 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 photodetector 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 5I are diagrams of a PCR device according to a fifth embodiment of the present invention.
  • the PCR device includes a heater group having one or more heaters, two or more heater groups, and the two or more heater groups are spaced apart so that mutual heat exchange does not occur.
  • An electrode unit having an electrode connected to supply electric power to heaters provided in the thermal block;
  • a PCR chip 10 according to the second 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 disposed 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 heaters 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, that is, the denaturation step, the annealing step and the extension step 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.
  • 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
  • in two steps for performing the PCR 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. to 80 ° C., preferably 72 ° 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 repeatedly provided sequentially.
  • 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 disposed, 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 is maintained by maintaining a temperature equal to or different from the temperature of the third heater 121c in the range of 80 ° C. so 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 in temperature can be greatly improved by repeatedly disposing two or more heaters maintaining a constant temperature.
  • the rate of change of the temperature between the heaters can be made 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. It doesn't happen.
  • 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 of 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.
  • FIG. 5E shows the structure of a PCR device according to a fifth embodiment of the present invention including the PCR chip 10. Specifically, the upper end of FIG. 5E shows a lateral cross-sectional view of the PCR chip 10 and the column block 100c of the PCR device according to the fifth embodiment of the present invention, and the lower end of FIG. 5E shows the fifth embodiment of the present invention.
  • the top plan view of the PCR chip 10 and the column block 100c of the PCR apparatus according to the example is shown.
  • the heat block 100c includes a heater unit that is repeatedly disposed ten times, and the heater unit includes a first heater group and a second heater group, and the first heater group and the second heater group. Each includes one heater, that is, the first heater 110c and the second heater 120c.
  • the heater, the heater group, the heater unit and the heat block according to FIG. 5E are as described above.
  • the electrode unit 200c is a module that receives power from a power supply unit (not shown) and supplies power to heat the heat block 100c, and supplies power to heaters provided in the heat block 100c. And electrodes 210c and 220c connected to each other. According to FIG. 5E, the first electrode 210c of the thermal block 100c is connected to supply power to the first heater 110c, and the second electrode 220c is connected to the second heater 120c. The electrode unit 200c is connected to supply power to the power source, but the electrode unit 200c may be disposed to be external to 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 210c may receive power from the power supply for maintaining the PCR denaturation step temperature
  • the second electrode 220c may receive power from the power supply for maintaining the PCR annealing / extension step temperature.
  • the first electrode 210c and the second electrode 220c may be connected to the first heater 110c and the two or more second heaters 120c that are repeatedly disposed in the column block 100c.
  • the first electrode 210c and the second electrode 220c may be conductive materials such as gold, silver, and copper, 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 includes an upper corresponding part 301c of the first heater 110c and an upper corresponding part of the second heater 120c disposed in the thermal block 100c.
  • 302c extends in fluid communication and extends in the longitudinal direction.
  • the upper corresponding portion 301c of the first heater 110c of the reaction channel 14 is a region where a PCR denaturation reaction of nucleic acid present in the target sample occurs, and the upper corresponding portion 302c of the second heater 120c. ) May be a region where PCR annealing / extension reactions of nucleic acids present in a target sample occur. That is, PCR is performed while the target sample flowing through the reaction channel 14 passes through the upper corresponding portion 301c of the first heater 110c and the upper corresponding portion 302c of the second heater 120c in succession. Can be.
  • the PCR chip 10 may include inlets 15 and outlets 16 at both ends of the one or more reaction channels 14, respectively. Meanwhile, as shown in the lower part of FIG.
  • the one or more reaction channels 14 are upper side of the upper side corresponding portion 301c of the first heater 110c disposed best and the second heater 120c disposed last. It is preferable to extend so that the corresponding part 302c may pass in a straight longitudinal direction. Therefore, the target sample introduced through the inlet 15 passes through the reaction channel 14 in the longitudinal direction, and the upper side of the upper portion 301c of the first heater 110c and the upper side of the second heater 120c.
  • the PCR denaturation step and the PCR annealing / extension step may be repeatedly performed and 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.
  • Figure 5f shows a PCR device according to a fifth embodiment of the present invention including a PCR chip, a power supply and a pump.
  • the PCR device includes a PCR chip 10, a power supply unit 400c, and a pump 500c.
  • the PCR chip 10 is disposed in contact on the heat block.
  • the PCR chip 10 and the components included therein are as described above.
  • the power supply unit 400c is a module for supplying power to the electrode unit 200c and may be connected to the first electrode 210c and the second electrode 220c of the electrode unit 200c, respectively.
  • a first power port (not shown) of the power supply 400c may be electrically connected to the first electrode 210c.
  • the second power port (not shown) of the power supply unit 400c is electrically connected to the second electrode 220c. Subsequently, when there is a user instruction for performing PCR, the power supply unit 400c supplies power to the first electrode 210c and the second electrode 220c, respectively, so that the first heater 110c and The second heater 120c may be quickly heated, and when the heaters 110c and 120c reach the predetermined temperature, the amount of power supplied is controlled to maintain the predetermined temperature.
  • the predetermined temperature may be a PCR denaturation step temperature (85 ° C to 105 ° C, preferably 95 ° C) in the first heater 110c and a PCR annealing / extension step temperature (in the second heater 120c). 50 ° C.
  • a PCR annealing / extension step temperature 50 ° C. to 80 ° C., preferably 72 ° C.
  • the second heater 120c In the PCR denaturation step temperature (85 °C to 105 °C, preferably 95 °C).
  • the pump 500c is a module for controlling the flow rate and the flow rate of the fluid flowing in the one or more reaction channels 14 of the PCR chip 10, and may be a positive pressure pump or a negative pressure pump, for example, a syringe It may be a syringe pump.
  • the pump 500c may be operably arranged in a portion of the reaction channel 14, but preferably the inlet 15 and / or outlet 16 formed at both ends of the reaction channel 303c. Is placed in the connection.
  • the pump 500c When the pump 500c is arranged to be connected to the inlet 15 and / or outlet 16, it serves as a pump as well as a target sample through the inlet 15 and / or outlet 16. It can also act as a stopper to prevent leakage of the solution.
  • the pump 500c may include one of the inlet 15 and the outlet 16. It can be connected to only one, and a general stopper can be sealedly connected to the remaining one, and the pump (if the flow rate and flow rate of the fluid flowing in the reaction channel 14, i. 500c may be connected to both the inlet 15 and the outlet 16.
  • the nucleic acid amplification reaction of the target sample in the PCR device including the PCR chip 10, the power supply 400c, and the pump 500c may be performed through the following steps.
  • oligonucleotide primer having a sequence complementary to the specific nucleotide sequence to be amplified
  • DNA polymerase DNA polymerase
  • dNTP deoxyribonucleotide triphosphates
  • PCR reaction buffer Prepare a target sample solution containing.
  • the target sample solution is introduced into the PCR chip 10.
  • the target sample solution is disposed in the reaction channel 13 inside the PCR chip 10 through the inlet 15.
  • the electrode portion 200c specifically, the first electrode 210c and the second electrode 220c are connected to the power supply 400c, respectively, and the inlet 14 of the PCR chip 10 And the outlet part 15 is sealingly connected to the pump 500c.
  • the power supply unit 400c is instructed to supply power to heat the first heater 110c and the second heater 120c through the first electrode 210c and the second electrode 220c, and A specific temperature is maintained, for example, the PCR denaturation step temperature (95 ° C.) for the first heater 110c and the PCR annealing / extension step temperature (72 ° C.) for the second heater 120c.
  • the target sample solution is supplied to the reaction channel 14 Allow it to flow horizontally from the inside.
  • the flow rate and flow rate of the target sample solution may be controlled by adjusting the intensity of the positive pressure or the negative pressure provided by the pump 500c.
  • the target sample solution is transferred from the inlet 15 end of the reaction channel 14 to the outlet 16 end of the upper corresponding portion 301c and the second of the first heater 110c.
  • PCR is performed while moving the upper corresponding portion 302c of the heater 120c in the longitudinal direction.
  • the first sample solution is supplied with heat from a heat block 100c in which a heater unit including the first heater 110c and the second heater 120c is repeatedly disposed 10 times. 10 PCR cycles are completed while undergoing a PCR denaturation step at the upper corresponding part 301c of 110c and a PCR annealing / extending step at the upper corresponding part 302c of the second heater 120c.
  • the target sample solution includes an upper corresponding portion 301c of the first heater 110c and the second heater from the outlet 16 end of the reaction channel 14 to the end of the inlet 15. PCR can be performed again while the upper correspondence part 302c of 120c is moved back in the longitudinal direction.
  • FIG. 5G illustrates a PCR device according to a fifth exemplary embodiment of the present invention including a PCR chip made of a light transmissive material and a heat block including a light source disposed between a first heater and a second heater, a power supply unit, a pump, and a light detector. Shows.
  • the PCR device includes a PCR chip 10 made of a light transmissive material and a light source 150c between the first heater 110c and the second heater 120c.
  • the arranged thermal block 100c includes a power supply unit 400c, a pump 500c, and a light detector 600c. The power supply and the pump are as described above.
  • the PCR chip 10 according to FIG. 5G is made of a light transmissive material, and the light source 150c is disposed between the first heater 110c and the second heater 120c of the thermal block 100c. .
  • the PCR apparatus further includes a light detector 600c for detecting the light emitted from the light source 150c.
  • the PCR device according to the fifth embodiment of the present invention according to FIG. 5G may measure and analyze the nucleic acid amplification process in real-time when performing PCR.
  • a separate fluorescent substance may be further added to the target sample solution, which induces a measurable and analytical light signal by emitting light with a specific wavelength according to the generation of the PCR product.
  • the light source 150c may be disposed in the spaced space between the first heater 110c and the second heater 120c and may emit the same light.
  • the light source 150c may be operably connected to a lens (not shown) that collects light emitted from the light source 150c and an optical filter (not shown) that filters light of a specific wavelength band.
  • the target sample solution may be PCR while successively passing the upper corresponding portion 301c of the first heater 110c and the upper corresponding portion 302c of the second heater 120c in the reaction channel 14.
  • a denaturation step and a PCR annealing / extension step are performed, in which case the target sample solution is between the first heater 110c and the second heater 120c, and the first heater 110c and the second heater 120c. Passes through the upper side corresponding to the light source 150c between the heater unit including.
  • the fluid control slows down or briefly stops the flow rate of the target sample solution and then emits light from the light source 150c, and emits the light.
  • the light is passed through the optically transparent PCR chip 10, specifically, the reaction channel 14, and the optical detector 600c measures and analyzes an optical signal generated by nucleic acid amplification in the reaction channel 14. can do. Therefore, the amount of target nucleic acid is monitored in real-time by monitoring the result of the reaction by amplification of nucleic acid (with fluorescent substance bound) in the reaction channel 14 during each cycle of PCR. -time) can be measured and analyzed.
  • FIG. 5H shows a PCR device according to a fifth embodiment of the present invention including a heat block made of a light transmissive material and a PCR chip, a power supply unit, a pump, a light providing unit, and a light detecting unit.
  • the PCR device includes a heat block 100c and a PCR chip 10, a power supply unit 400c, a pump 500c, and a light provider 700c of a light transmissive material. , And the light detector 800c.
  • the power supply 400c and the pump 500c are as described above.
  • the PCR device of FIG. 5H is characterized in that the thermal block 100c and the PCR chip 10 are implemented with a light transmissive material.
  • the PCR apparatus further includes a light providing unit 700c arranged to provide light to the PCR chip 10 and a light detecting unit 800c arranged to receive light emitted from the PCR chip 10. . Therefore, the PCR device according to the fifth embodiment of the present invention can measure and analyze the nucleic acid amplification process in real-time when performing PCR.
  • a separate fluorescent substance may be further added to the target sample solution, which induces a measurable and analytical light signal by emitting light with a specific wavelength according to the generation of the PCR product.
  • the heat block 100c of the light transmissive material 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.
  • a part of the excitation light may be reflected on the heat block 100 of the light transmissive material or after passing through the heat block 100c of the light transmissive material to act as noise of the optical signal. Therefore, the light absorbing material may be formed on the lower surface of the heat block 100c of the light transmissive material to form a light absorbing layer (not shown), thereby further increasing the sensing efficiency.
  • 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 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 prevention layer (not shown) may be formed on the upper surface of the heat block 100c of the light transmissive material to further increase the sensing efficiency.
  • 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 processed on the lower surface of the heat block 100c of the light transmissive material, and at the same time, the light reflection preventing material is treated on the upper surface of the heat block 100c of the light transmissive material for sensing
  • the efficiency can be further increased. That is, in order to effectively monitor real-time PCR, 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.
  • the reflectance of the excitation light of the conventional heaters of a general metallic material is about 20% to 80%, but the light reflectance is 0.2 when using the heat block 100c of the light transmissive material including the light absorption layer or the light reflection prevention layer.
  • the light reflectance can be reduced to less than or equal to 4%, and when the heat block 100c of the light transmissive material including the light absorbing layer and the light reflection prevention layer is used, the light reflectance can be reduced to 0.2% or less.
  • the light providing unit 700c is a module for providing light to the PCR chip 10 made of the light transmissive material, and the light detector 800c receives the light emitted from the PCR chip 10 made of the light transmissive material.
  • Light is emitted from the light providing part 700c, and the emitted light passes or reflects through the PCR chip 10 of the light transmissive material, specifically, the reaction channel 14, and in this case, the reaction channel 14
  • the optical detection unit 800c measures and analyzes an optical signal generated by nucleic acid amplification in the C).
  • the reaction result by amplification of nucleic acid (phosphorescent material bound) in the reaction channel 14 during each cycle of the PCR in the light transmitting material PCR chip 10 is real-time.
  • the target nucleic acid can be amplified and amplified in real time.
  • the light providing unit 700c and the light detecting unit 800c may be disposed above or below the heat block 100c of the light transmitting material.
  • the arrangement of the light providing unit 700c and the light detecting unit 800c may vary in consideration of an arrangement relationship with other modules for optimal implementation of the PCR apparatus.
  • the light providing unit 700c and The light detector 800c may be disposed above the thermal block 100c of the light transmissive material.
  • the light providing unit 700c may include a light emitting diode (LED) light source or a laser light source, a first light filter for selecting light having a predetermined wavelength from light emitted from the light source, and light emitted from the first light filter. And a first optical lens configured to collect light, and further comprising a first aspherical lens disposed to spread light between the light source and the first light filter.
  • the light source includes all light sources capable of emitting light, and according to an embodiment of the present invention, includes a light emitting diode (LED) light source or a laser light source.
  • the first light filter 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.
  • the first light filter may pass only light in a wavelength band of 500 nm or less of the light emitted from the light source.
  • the first optical lens collects the incident light and increases the intensity of the emitted light, and the intensity of light irradiated onto the PCR chip 10 of the light transmissive material through the heat block 100c of the light transmissive material. Can be increased.
  • the light providing unit further includes a first aspherical lens disposed to spread light between the light source and the first light filter. By adjusting the arrangement direction of the first aspherical lens, the light range emitted from the light source is extended to reach the measurable area.
  • the light detector 800c may select a second optical lens for collecting light emitted from the PCR chip 10 made of the light transmissive material, and a second light for selecting light having a predetermined wavelength from the light emitted from the second optical lens.
  • a second aspherical lens disposed between the second aspherical lens and the optical analyzer to remove noise of light emitted from the second aspherical lens and to amplify the light emitted from the second aspherical lens. It may further include a photodiode integrated circuit (PDIC) (not shown).
  • PDIC photodiode integrated circuit
  • the second optical lens collects the incident light and increases the intensity of the emitted light, and the second optical lens collects the light emitted from the PCR chip 10 of the light transmitting material through the heat block 100c of the light transmitting material.
  • the intensity is increased to facilitate optical signal detection.
  • the second light filter selects and emits light having a specific wavelength among incident light having various wavelength bands, and is predetermined from the PCR chip 10 of the light transmissive material through the heat block 100c of the light transmissive material. It may be variously selected according to the wavelength of light.
  • the second light filter may pass only light in a wavelength range of 500 nm or less among predetermined light emitted from the PCR chip 10 of the light transmissive material through the heat block 100 of the light transmissive material. .
  • the optical analyzer is a module for detecting an optical signal from the light emitted from the second optical filter, and converts the fluorescent light expressed from the target sample solution into an electrical signal to enable qualitative and quantitative measurement.
  • the light detector 800c may further include a second aspherical lens disposed between the second light filter and the light analyzer to integrate light emitted from the second light filter. By adjusting the arrangement direction of the second aspherical lens, the light range emitted from the second light filter is extended to reach the measurable area.
  • the light detector 800c is disposed between the second aspherical lens and the optical analyzer to remove noise of light emitted from the second aspherical lens and to amplify the light emitted from the second aspherical lens. It may further include a photodiode integrated circuit (PDIC). By using the photodiode integrated device, the PCR device can be further miniaturized, and noise can be minimized to measure a reliable optical signal.
  • PDIC photodiode integrated circuit
  • the PCR apparatus may adjust one or more dichroic filters (750x) for adjusting light propagation direction so that light emitted from the light providing part 700c may reach the light detecting part 800c and separating light having a predetermined wavelength. 750y).
  • the dichroic filters 750x and 750y are modules that reflect light at an angle that is selectively transmitted or selectively adjusted according to the wavelength.
  • the dichroic filter 750x 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 700c, and selectively transmits the short wavelength component and reflects the long wavelength component at right angles according to its wavelength. Reach the PCR chip 10 of the light transmissive material disposed on the heat block 100c of the light transmissive material.
  • the dichroic filter 750y 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 thermal block 100c of the light transmissive material, and selectively shifts the light according to its wavelength.
  • the short wavelength component is transmitted and the long wavelength component is reflected at right angles to reach the light detector 800c.
  • the light reaching the light detector 800c is converted into an electrical signal in the optical analyzer to indicate whether the nucleic acid is amplified and the degree of amplification.
  • the target sample solution may be PCR while successively passing the upper corresponding portion 301c of the first heater 110c and the upper corresponding portion 302c of the second heater 120c in the reaction channel 14.
  • a denaturation step and a PCR annealing / extension step are performed.
  • the reaction from the light provider 700c may be slowed down or briefly stopped through the fluid control, and then the reaction may be performed.
  • the light detector 800c may receive light at an arbitrary position in the channel 14 and receive the light emitted by the amplification reaction of the target nucleic acid in the reaction channel 14 to measure and analyze the optical signal.
  • the light providing unit 700c and the light detecting unit 800c are arranged in pairs, but in order to measure and analyze a plurality of optical signals generated at arbitrary positions in the reaction channel 14. It can be arranged in pairs at appropriate locations. Therefore, the amount of target nucleic acid is monitored in real-time by monitoring the result of the reaction by amplification of nucleic acid (with fluorescent substance bound) in the reaction channel 14 during each cycle of PCR. -time) can be measured and analyzed.
  • Pathogenic microorganisms causing five food poisonings were purchased from Korea Microbial Conservation Center, Korea Biomass Center, and The global bioresource center. After culturing the pathogenic microorganisms, food poisoning genomic DNA was extracted using a QiaAmp DNA Purification Kit (QIAGEN).
  • EIEC Escher coli
  • Campylobacter coli is the ceuE gene
  • the gene targeted in Vibrio cholerae is the ompW gene
  • the gene targeted in Vibrio vulnificus is the Vvh gene.
  • a genomic DNA of the food poisoning bacterium obtained in Experimental Example 1 was used as a template, and the primer set for detecting food poisoning bacteria according to one embodiment of the present invention described in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, Assuming that SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10), a PCR device of another company (BIO-RAD) and a PCR device according to a third embodiment of the present invention Real time PCR was performed.
  • the composition of the reaction solution for real-time PCR and the reaction conditions of real-time PCR are described in Tables 2 and 3 below, and the PCR device according to the third embodiment of the present invention.
  • the composition of the reaction solution for the real-time PCR and the reaction conditions of the real-time PCR are described in Tables 4 and 5 below.
  • 6A and 6B are electrophoresis images showing real-time PCR results of a sample using a PCR device of another company and a PCR device according to a third embodiment of the present invention, respectively.
  • number 1 is a negative control
  • number 2 is a primer set of SEQ ID NO: 3 and SEQ ID NO: 4
  • number 3 is a primer set of SEQ ID NO: 1 and SEQ ID NO: 2
  • number 4 Is a primer set of SEQ ID NO: 5 and SEQ ID NO: 6
  • the number 5 is the primer set of SEQ ID NO: 7 and SEQ ID NO: 8
  • the number 6 shows the result by the primer set of SEQ ID NO: 9 and SEQ ID NO: 10.
  • Figure 6a and 6b when compared with the results of the PCR device of the other company, it can be confirmed that the food poisoning gene gene was detected accurately and quickly when using the primer set and the PCR device according to an embodiment of the present invention.
  • FIGS. 7A and 7B are graphs showing real-time PCR results of a sample using a third-party PCR device and a PCR device according to a third embodiment of the present invention, respectively. According to FIGS. 7A and 7B, it can be confirmed that when the primer set and the PCR device according to one embodiment of the present invention are used, the food poisoning bacteria genes are accurately and quickly detected in real time when compared with the results of other companies' PCR devices. (Table 6 below shows the Ct values of real time PCR for each number).
  • Table 6 number Primer set Third Party PCR Device (Ct value) PCR device (Ct value) according to a third embodiment of the present invention

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Abstract

La présente invention concerne un ensemble d'amorces pour la détection d'une intoxication alimentaire, un appareil de PCR à l'aide de celui-ci, et un procédé de détection d'une intoxication alimentaire à l'aide de ceux-ci, la présente invention permettant une détermination précise et rapide de l'infection à partir de bactéries alimentaires à un coût économique, empêchant ainsi la diffusion des bactéries alimentaires, et contribuant significativement à prendre des mesures de réponse rapide contre celle-ci.
PCT/KR2013/012237 2012-12-27 2013-12-27 Ensemble d'amorces pour la détection d'une intoxication alimentaire, appareil de pcr à l'aide de celui-ci, et procédé de détection d'une intoxication alimentaire à l'aide de ceux-ci WO2014104770A1 (fr)

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KR1020120154563A KR102028381B1 (ko) 2012-12-27 2012-12-27 식중독 검출용 프라이머 세트를 이용한 pcr 장치, 및 이를 이용한 식중독 검출 방법
KR10-2012-0154563 2012-12-27

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KR101971633B1 (ko) * 2017-07-24 2019-04-24 한국과학기술원 유전자 판별칩용 믹서장치 및 이를 이용한 pcr버퍼와 유전자 혼합방법
KR101971631B1 (ko) * 2017-07-24 2019-04-24 한국과학기술원 전자동 유전자 증폭기

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