KR20160110574A - Multiplex pcr chip and multiplex pcr device comprising the same - Google Patents

Abstract

According to one embodiment of the present invention, a multiplex PCR chip and a multiplex PCR apparatus including the same are provided. A plurality of probes for hybridization reaction that are hybridized specifically to different sequences of the nucleic acid molecules so as to simultaneously detect a plurality of nucleic acid molecules that are different from each other and are disposed apart from each other; And a plurality of probe probes disposed on an inner surface of the multiplex PCR chip to form a pore structure so as to increase a contact area between the probes and the nucleic acid molecules, Wherein the probe is characterized in that a fluorescent substance and a fluorescence inhibiting substance are bound to the terminal or middle of the base sequence, respectively.

Description

MULTIPLEX PCR CHIP AND MULTIPLEX PCR DEVICE COMPRISING THE SAME BACKGROUND OF THE INVENTION 1. Field of the Invention [0001]

The present invention relates to a multiplex PCR chip and a multiplex PCR apparatus including the same. More particularly, the present invention relates to a multiplex PCR chip for simultaneously detecting a plurality of mutually different nucleic acid molecules based on positions of a plurality of probes, To a multiplex PCR apparatus.

Polymerase Chain Reaction (PCR) is a method for repeatedly heating and cooling a sample solution containing a nucleic acid to successively replicate a region having a specific nucleotide sequence of the nucleic acid, As a technique of amplifying in a series, specifically, a series of temperature enzymatic reaction steps such as denaturation, annealing, and extension may be performed. PCR is widely used for analysis and diagnosis in life sciences, genetic engineering and medical fields.

On the other hand, the above-described diagnosis through nucleic acid amplification or the search for a specific gene has a limitation in that it searches for one template at a time. It is cumbersome and time consuming to amplify each template one template at a time in situations where you need to amplify several templates. For example, even if the same symptoms occur in the same patient, the cause of the onset is often due to various types of infectious agents, and diagnosis of various pathogens is needed individually. In addition, cancer and genetic defects are known to be caused by complex mutations of several genes. Polymorphism or mutation requires the examination of additional zygotes due to loci changes in various genes. Since the amount of nucleic acid that can be extracted from a limited sample in a general environment is limited, repetitive diagnosis using nucleic acid amplification using a limited amount of nucleic acid is often impossible.

Therefore, a technique for analyzing nucleic acids of many templates from the same sample is required, and this analysis technique can be referred to as multiplex PCR. In this regard, Figure 1 illustrates an exemplary process of conventional multiplex PCR.

Referring to FIG. 1, conventional multiplex PCR can perform PCR by injecting a plurality of primer sets into one reaction vessel (or tube). Multiple sets of primers can be specifically hybridized to various sequences of nucleic acid molecules, and thus multiple target nucleic acid sequences can be amplified simultaneously. In other words, multiplex PCR can detect and diagnose a plurality of genes and diseases in a single experiment, thereby reducing the number of experiments and labor, and providing a cost saving effect.

However, in order to monitor the amplification products of multiplex PCR in real time, special detection equipment is required, which increases the overall size and complexity of the PCR device and, consequently, it can be cost-uneconomical. Specifically, the monitoring of the amplification products of the multiplex PCR can be performed by irradiating the excitation light during the amplification reaction and detecting the emission light therefrom, Oligonucleotides (i.e., primers or probes) labeled with fluorescent dyes capable of generating a signal indicative of the presence of a target nucleic acid sequence during the reaction are used, particularly in multiplex PCR, to identify a number of different nucleic acid sequences that can be amplified Various oligonucleotides specific for each nucleic acid sequence may be used. That is, in conventional multiplex PCR, for detection of multiple target nucleic acid sequences, a plurality of fluorescent dyes must be labeled, and in order to detect multiple types of fluorescence from various types of fluorescent dyes, There is a need for light sources and filters of multiple wavelengths that are optimized for the detection of fluorescent dyes. This requires multiple wavelengths of measurement time to increase the time required to detect the nucleic acid sequence, increase the overall size and complexity of the PCR device, and consequently be cost-uneconomical.

Therefore, there is a need for a multiplex PCR apparatus that can simplify the entire structure, minimize the total PCR reaction time, and obtain a reliable PCR reaction yield.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a multiplex PCR apparatus for simultaneously detecting a plurality of nucleic acid molecules different from each other based on positions of a plurality of probes.

The technical problems of the present invention are not limited to the above-mentioned technical problems, and other technical problems which are not mentioned can be understood by those skilled in the art from the following description.

According to one embodiment of the present invention, a multiplex PCR chip is provided. The chip

A plurality of probes for hybridization reaction that are specifically hybridized with different sequences of the nucleic acid molecules so as to simultaneously detect a plurality of nucleic acid molecules that are different from each other and are spaced apart from each other; And

A plurality of probe coupling parts disposed on an inner surface of the multiplex PCR chip to form a pore structure so as to increase a contact area between the probe and the nucleic acid molecule so that the probes are respectively coupled to the porous structure; ≪ / RTI &

The probe may be characterized in that a fluorescent substance and a fluorescence inhibiting substance are respectively bound to the terminal or middle of the base sequence.

According to one embodiment of the present invention, a multiplex PCR apparatus is provided. The apparatus comprising: the multiplex PCR chip; A light supplier for irradiating an excitation light toward the probe in the multiplex PCR chip; And a photodetector for detecting an emission light generated in the plurality of probes by the excitation light, wherein the detection by the photodetector and the photodetector is performed using light of a single wavelength or a plurality of wavelengths .

According to one embodiment of the present invention, a multiplex PCR apparatus is provided. The apparatus comprising: the multiplex PCR chip; And at least one column block for contacting the multiplex PCR chip and transferring heat for multiplex PCR to the multiplex PCR chip.

According to the present invention, by disposing a plurality of probes that are specifically hybridized with sequences of nucleic acid molecules that are mutually different from each other, the sequence of the nucleic acid molecules hybridized by the probes can be distinguished based on the positions of the probes, The need for different fluorescent dyes for labeling can be eliminated.

In addition, according to the present invention, multiplex PCR real-time PCR using a single fluorescent dye is possible because the sequence of the nucleic acid molecule hybridized with the probe can be distinguished based on the spacing between the probes. This makes it possible to miniaturize the size of the optical equipment and reduce the equipment cost by using only one kind of light source and filter, and to improve the operation efficiency of the multiplex PCR apparatus by reducing the time required for detection have.

Further, according to the present invention, a plurality of probes can be bonded on a surface of a multiplex PCR chip through a predetermined probe coupling portion, thereby providing a more rigid bonding force, which can be obtained by separation and hybridization of the binding and a distorted result Can be prevented.

In addition, according to the present invention, the probe-coupled portion can form a pore structure, and the probe is bonded to the surface of the porous structure, thereby increasing the contact area between the probe and the multiplex PCR product, .

BRIEF DESCRIPTION OF THE DRAWINGS A brief description of each drawing is provided to more fully understand the drawings recited in the description of the invention.
Figure 1 illustrates an exemplary process of conventional multiplex PCR.
Figure 2 shows a multiplex PCR chip according to one embodiment of the present invention.
Figure 3 shows a multiplex PCR chip according to one embodiment of the present invention.
Figure 4 shows a multiplex PCR chip according to one embodiment of the invention.
Figure 5 shows a multiplex PCR device according to an embodiment of the present invention.
6A and 6B illustrate a multiplex PCR apparatus according to an embodiment of the present invention.
Figure 7 shows a multiplex PCR device according to an embodiment of the present invention.
Figure 8 shows the production of a multiplex PCR chip according to an embodiment of the present invention.
FIGS. 9 to 11 show the results obtained by the experimental example according to the embodiment of the present invention.

Hereinafter, embodiments according to the present invention will be described with reference to the accompanying drawings. It should be noted that, in adding reference numerals to the constituent elements of the drawings, the same constituent elements are denoted by the same reference symbols as possible even if they are shown in different drawings. In the following description of the embodiments of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the difference that the embodiments of the present invention are not conclusive. In addition, embodiments of the present invention will be described below, but the technical idea of the present invention is not limited thereto and can be variously modified by those skilled in the art.

Throughout the specification, when a part is referred to as being "connected" to another part, it includes not only "directly connected" but also "indirectly connected" . Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise. In describing the components of the embodiment of the present invention, terms such as first, second, A, B, (a), and (b) may be used. These terms are intended to distinguish the constituent elements from other constituent elements, and the terms do not limit the nature, order or order of the constituent elements.

The multiplex PCR apparatus according to the present invention is an apparatus for performing multiplex PCR (Multiplex Polymerase Chain Reaction) for amplifying various nucleic acids having a specific base sequence. Specifically, in order to amplify deoxyribonucleic acid (DNA) having a specific nucleotide sequence, a multiplex PCR apparatus is constructed by heating a sample solution containing double stranded DNA at a specific temperature, for example, about 95 ° C, A denaturing step of separating into a single strand of DNA and an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified in the sample solution, An annealing step of cooling the solution to 55 ° C to bind a primer to a specific nucleotide sequence of single stranded DNA to form a partial DNA-primer complex, and a step of annealing the sample solution at an appropriate temperature, for example, And maintained at 72 ° C to form a double-stranded DNA based on a primer of a partial DNA-primer complex by a DNA polymerase DNA having a specific nucleotide sequence can be exponentially amplified by performing an extension step (extension step) and repeating step 3, for example, 20 to 40 times. In some cases, the PCR apparatus can simultaneously perform the annealing step and the extension (or amplification) step. In this case, the PCR apparatus performs two steps consisting of an extension step and an annealing and extension (or amplification) 1 cycle may be completed. Accordingly, a multiplex PCR apparatus according to an embodiment of the present invention refers to an apparatus including modules for performing steps, and the detailed modules not described herein are disclosed in the prior art for performing PCR, Are all included in the range.

In addition, the multiplex PCR apparatus according to the present invention can perform multiplex PCR and simultaneously measure the presence or absence of the multiplex PCR product and measure it in real time.

Figure 2 shows a multiplex PCR chip according to one embodiment of the present invention.

Referring to FIG. 2, a multiplex PCR chip 200 is provided for performing amplification (amplification reaction) of a nucleic acid molecule, detection (hybridization reaction) of a target sequence, and the like, in which one or more reaction regions 224 . The fluid may be a nucleic acid, for example, a double stranded DNA, an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, a DNA polymerase, deoxyribonucleotide triphosphates (dNTP), a PCR reaction buffer reaction buffer) and the like.

At least a portion of the multiplex PCR chip 200 may be implemented with a light transmissive material, and preferably a light transmissive plastic material. The multiplex PCR chip 200 can increase the heat transfer efficiency only by adjusting the thickness of the plastic using the plastic material, and the manufacturing process can be simplified, thereby reducing the manufacturing cost. Since the multiplex PCR chip 200 can have a light transmitting property as a whole, the multiplex PCR chip 200 can be directly irradiated with light in a state where it is disposed on one side of the thermal block, can do. For amplification reactions, when the multiplex PCR chip 200 contacts a thermal block, the heat block sequence is transferred to the multiplex PCR chip 200 and included in the reaction region 224 of the multiplex PCR chip 200 The fluid may be heated or cooled to maintain a constant temperature. The multiplex PCR chip 200 preferably has a generally planar shape, but is not limited thereto.

As shown in FIG. 2, the multiplex PCR chip 200 may include a probe 240 disposed therein for hybridization reaction. The probe 240 may be specifically hybridized with a sequence of a nucleic acid molecule as a labeled oligonucleotide capable of generating a signal indicative of the presence of a target nucleic acid sequence during an amplification reaction to detect a nucleic acid amplified through PCR. Each probe 240 can be hybridized to a different sequence of nucleic acid molecules.

In particular, the probe 240 may be (shared) conjugated to, for example, a portion of the base sequence, that is, a fluorophore and a fluorescence quencher, respectively, at the end or middle of the base sequence. Here, the fluorescent substance refers to a fluorescent substance such as FAM, and the fluorescent substance inhibits the fluorescence of the fluorescent substance through, for example, FRET (fluorescence resonance energy transfer). For example, BHQ-1 (black hole quencher-1), and the like. According to the probe 240 having such a structure, in the annealing step during the PCR reaction, the probe 240 is specifically hybridized to the sequence of the nucleic acid molecule (that is, the template DNA), wherein the fluorescence of the fluorescent substance Generation is suppressed. However, the probe 240 is degraded due to the activity of exonuclease possessed by the DNA polymerase in the prolonged phase of the PCR reaction. In this case, since the fluorescent substance is liberated from the probe, Lt; / RTI > That is, the probe 240 can detect the target nucleic acid (or the target nucleic acid sequence) through fluorescence through hybridization with the target nucleic acid sequence, free and distant.

Further, each of the probes 240 can be coupled to each other on the surface of the multiplex PCR chip 200. [ This coupling is performed by applying the probes 240 on the surface of the multiplex PCR chip 200 using, for example, a spotter, an arrayer, an ink-jet, or the like . By detecting the position of the fluorescence through the spacing between the probes, the nucleic acid to be detected can be identified.

Each of the probes 240 may be adsorbed on the surface of the multiplex PCR chip 200, or may be coupled through the probe coupling portion. The probe coupling portions can provide a stronger bonding force than that of the conventional probe 240 and the multiplex PCR chip 200. [ The probes 240 may be coupled to the surface of the porous structure so that the contact between the probe 240 and the multiplex PCR product (i.e., the amplified nucleic acid molecule) By increasing the area, the reactivity can be improved. The size of the porous structure depends on the molecular weight, UV curing and washing conditions, and the size thereof can be formed from the nanometer level to the micrometer level, thereby controlling the reactivity between the porous structure and the multiplex PCR product . The porous structure may be selected from the group consisting of polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDMA), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol But are not limited to, ethylene glycol diacrylate (EGDA), ethylene glycol dimethacrylate (EGDMA), polyvinyl alcohol (PVA), agarose, silicone, and paraffin. Forming material (porogen) composed of at least one of gel material, polyethylene glycol (PEG), and ethylene glycol (EG) composed of at least one of a photoinitiator and a buffer have. The photoinitiator may be a linker or spacer such as a PEG linker, a C linker, a TEG linker or the like, a 2-hydroxy-2-methyl- 1-phenyl-1-propanone, methylbenzoylformate, and the buffer may be a Tris-EDTA buffer. In this case, the molecular weight of polyethylene glycol diacrylate (PEGDA) may be 10 to 50,000 MW, the molecular weight of polyethylene glycol (PEG) may be 10 to 50,000 MW, and the content of polyethylene glycol diacrylate Polyethylene glycol diacrylate (PEGDA) has a concentration ratio of 5 to 40%, polyethylene glycol (PEG) has a weight ratio of 5 to 40%, 2-hydroxy- 2-methyl-1-phenyl-1-propanone) may have a concentration ratio of 1 to 10% and a Tris-EDTA buffer may have a concentration ratio of 0.1 to 30%. Meanwhile, the probe-binding unit may include a linker material such as 18-atom hexa-ethleneglycol or an acrydite binding material thiol (binding agent) such as hexa-ethylene glycol A porous structure binding material such as a thiol binding material, a biotin binding material, and an amine binding material. In addition, the probe-binding unit may further include a plurality of primers that specifically hybridize with different sequences of the nucleic acid molecule, so that the nucleic acid molecule hybridizes with the probe after hybridizing to the primer. The probe 240 may also be disposed on the top surface of the reaction zone 224 (or the top surface of the multiplex PCR chip 200 or the bottom surface of the third plate 230). During the PCR reaction, bubbles may be generated, which may cause interference in measuring the PCR reaction product, but as shown in FIG. 2, the probe 240 is disposed on the upper surface of the reaction zone 224, The bubbles are moved to the vicinity of the probe 240, and thus the interference can be removed to improve the measurement efficiency.

The same fluorescent dyes may be used for the plurality of probes 240. In order to distinguish the sequence of the nucleic acid molecules hybridized by the plurality of probes 240 in the conventional multiplex PCR, the probes 240 labeled with fluorescent dyes having different colors should be used, A plurality of probes 240 are arranged at predetermined intervals so that the sequence of the nucleic acid molecules hybridized by the probe 240 can be distinguished based on the position between the probes 240 even if the same fluorescent dye is used , The need for different fluorescent dyes can be eliminated.

Use of such the same fluorescent dye can simplify an optical apparatus for detecting fluorescence by a fluorescent dye. In conventional multiplex PCR, a plurality of different probes 240, which can be specifically hybridized with amplified sequences of nucleic acid molecules in one reaction vessel, are labeled with different fluorescent dyes, and fluorescence an optical device having a plurality of wavelengths specific to each fluorescent dye was used. However, according to the present invention, excitation light having a single wavelength is directed toward a plurality of probes 240, so that even if fluorescence by the same dye sample, that is, fluorescence of the same color is generated, The sequence of the nucleic acid molecule to be amplified can be distinguished. That is, in the present invention, by using one kind of light source and filter, it is possible to detect a multiplex PCR product, which can miniaturize the optical equipment and reduce the equipment cost, The efficiency of operation of the multiplex PCR apparatus can be improved. However, the excitation light having a plurality of wavelengths can be irradiated toward the plurality of probes 240 according to the purpose of the user, and the sequence of the nucleic acid molecules amplified based on the position between the probes 240 can be distinguished, It is possible to more accurately perform a plurality of measurements, thereby maximizing the efficiency of operation of the multiplex PCR apparatus.

The structure of the multiplex PCR chip 200 shown in FIG. 2 will be described in detail. A first plate 210 having a flat plate shape may be provided as a base of the multiplex PCR chip 200. The second plate 220 and the third plate 230 may be sequentially disposed on the first plate 210. [ The second plate 220 may be disposed on the first plate 210. The second plate 220 includes an inlet 222 through which a fluid (e.g., a sample solution containing a nucleic acid to be amplified) flows, a reaction region where the introduced fluid moves and a PCR reaction and a hybridization reaction are performed 224, and an outlet 226 through which the reacted fluid is discharged. As shown, the reaction zone 224 of the second plate 220 is recessed from the surface (e.g., the upper surface and / or the lower surface) of the second plate 220, . The inlet portion 222 and the outlet portion 226 of the second plate 220 may protrude from the surface of the second plate 220 while passing through the second plate 220. In addition, the thickness of the second plate 220 may vary, but may be selected from 0.1 mm to 2.0 mm. The width and length of the reaction zone 224 may vary but preferably the width of the reaction zone 224 is selected from 0.5 mm to 3 mm and the length of the reaction zone 224 is from 20 mm to 60 mm ≪ / RTI > The inner wall of the second plate 220 may be coated with a material such as silane series or bovine serum albumin (BSA) to prevent adsorption of DNA and protein, The treatment can be carried out according to methods known in the art. In addition, the inlet 222 may have various sizes, but may preferably be selected from 0.5 mm to 3.0 mm in diameter. In addition, outlet 226 may have varying sizes, but may preferably be selected from 0.5 mm to 3.0 mm in diameter. The third plate 230 may be disposed on the second plate 220. Specifically, the third plate 230 is disposed on the second plate 220, and a part of the reaction area 224 of the second plate 220 (i.e., the reaction area 224 of the second plate 220 And at least one probe 240 spaced apart from each other on a partial area of the lower surface of the third plate 230. The PCR reaction product can be measured using the probe 240. [ The thickness of the third plate 230 may vary, but may preferably be selected from 0.1 mm to 2.0 mm. At least one of the first plate 210, the second plate 220 and the third plate 230 may be formed by injection molding, hot-embossing, casting, laser ablation, And the like can be formed by various mechanical and chemical processing processes. The above-described processing steps are illustrative, and various processing steps can be applied according to the embodiment to which the present invention is applied. The joining between the first plate 210 and the second plate 220 and / or the joining between the second plate 220 and the third plate 230 may be performed by, for example, thermal bonding, ultrasonic joining, ultraviolet joining, Bonding, tape bonding, and the like can be performed by various bonding methods applicable in the art. Depending on the embodiment, at least some of the inner surfaces of the multiplex PCR chip 200 (e.g., the inner walls of the second plate 220, etc.) may be subjected to a surface treatment. For example, it may be coated with a substance such as silane series, bovine serum albumin (BSA) or the like to prevent adsorption of DNA and protein on the surface, May be performed according to various techniques known in the art. Also, according to an embodiment, the multiplex PCR chip 200 is provided with separate cover means (not shown) for the inlet 222 and / or the outlet 226, so that the inlet 222 and outlet It is possible to prevent contamination of the inside of the multiplex PCR chip 200 through the portion 226 or to prevent leakage of fluid injected into the microfluidic chip 200. [ Such cover means can be embodied in various shapes, sizes or materials. The shape and structure of the multiplex PCR chip 200 shown in FIG. 2 are illustrative, and microfluidic chips of various shapes and structures can be used according to the embodiment to which the present invention is applied. The first plate 210, the second plate 220, or the third plate 230 may be formed of various materials, but it is preferable to use polymethylmethacrylate (PMMA), polycarbonate , PC), cycloolefin copolymer (COC), polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE) (PS), polyoxymethylene (POM), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), polydimethylsiloxane (PDMS), polypropylene carbonate (PPC), polyether sulfone (PES), polyethylene terephthalate (PET), polypropylene carbonate (PPC), polyether sulfone Polyether sulfone (PES), and combinations thereof.

Figure 3 shows a multiplex PCR chip according to one embodiment of the present invention.

Referring to FIG. 3, in the multiplex PCR chip 300, the third plate 230 is disposed in a portion of the reaction region 224 of the second plate 220 (that is, the reaction region of the second plate 220 224). For this, a part of the lower surface of the third plate 230 may protrude downward. The protruding region covers the penetration region of the reaction region 224 of the second plate 220 and facilitates insertion of the third plate 230 and the second plate 220 through the insertion into the penetration region, A junction alignment can be achieved. The shape of the multiplex PCR chip 300 shown in FIG. 3 is illustrative and various shapes can be applied according to the embodiment to which the present invention is applied. On the other hand, the hydrophilic material 310 is processed in at least one region of the inner surface of the multiplex PCR chip 300 (that is, one region of the lower surface of the third plate 230) so that the multiplex PCR can be performed smoothly have. The hydrophilic material 310 may be various materials but is preferably selected from the group consisting of a carboxyl group (-COOH), an amine group (-NH2), a hydroxyl group (-OH), and a sulfone group (-SH) . In addition, the treatment of the hydrophilic material 310 may be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application, but this is illustrative only, , Various treatment methods known in the art can be applied.

Figure 4 shows a multiplex PCR chip according to one embodiment of the invention.

4 (a) is a plan view of the multiplex PCR chip 500, FIG. 4 (b) is a cross-sectional view of the multiplex PCR chip 500 in the AA 'direction, (c) shows a bottom perspective view of the inner surface of the multiplex PCR chip 500 shown in Figs. 4 (a) and 4 (b).

Referring to FIG. 4, the multiplex PCR chip 500 may further include a probe fixing unit 510. The probe fixing unit 510 is for receiving and fixing the probe 240 for detecting the target sequence and may be formed in one area of the lower surface of the third plate 230 of the multiplex PCR chip 500 And a peripheral portion 514 protruding so as to surround the central portion 512. The center portion 512 may provide a space for accommodating the probe 240 and the peripheral portion 514 may prevent the probe 240 accommodated in the center portion 512 from detaching.

The shape of the probe fixing part 510 shown in FIG. 4 is an exemplary one, and probe fixing parts having various shapes can be used according to the embodiment to which the present invention is applied.

Figure 5 shows a multiplex PCR device according to an embodiment of the present invention.

5, a multiplex PCR apparatus 1000 includes a thermal block 900, multiplex PCR chips 200 to 700, optical fibers (not shown) operably arranged to provide light to the multiplex PCR chips 200 to 700, And may further include a photodetector portion 1020 which is arranged to be capable of receiving light emitted from the multiplexer PCR chips 200 to 700 and the optical fiber 1010.

The optical supplier 1010 may be a module for providing light to the multiplex PCR chips 200 to 700. In one embodiment, the optical fiber 1010 includes a light source that emits light, such as a light emitting diode (LED) light source or a laser light source, a first optical filter that selects light having a predetermined wavelength from light emitted from the light source, And a first optical lens that collects the light emitted from the first optical filter and increases the intensity of the emitted light. According to an additional embodiment, the optical providing portion 1010 may further include a first aspherical lens arranged to spread light between the light source and the first optical filter. That is, by adjusting the arrangement direction of the first aspherical lens, it is possible to extend the light range emitted from the light source to reach the measurable area. However, the configuration of the optical distributor 1010 is not limited thereto.

The optical detector 1020 is a module for measuring the PCR reaction product performed in the multiplex PCR chips 200 to 700 by receiving light emitted from the multiplex PCR chips 200 to 700. The optical detector 1020 includes a light supplier 1010, Is passed or reflected by the multiplex PCR chip 200 to 700, specifically the reaction region 224 of the multiplex PCR chip 200 to 700 or the probe 240, and in this case is generated by nucleic acid amplification The optical detecting unit 1020 can detect the optical signal. In one embodiment, the photodetector 1020 includes a second optical lens that collects light emitted from the multiplex PCR chips 200 to 700 and increases the intensity of the emitted light, And an optical analyzer for detecting optical signals from the light emitted from the second optical filter. According to a further embodiment, a second aspheric lens arranged to integrate the light emitted from the second optical filter between the second optical filter and the optical analyzer, and / or a second aspherical lens between the second aspherical lens and the optical analyzer And a photodiode integrated circuit disposed to remove noise of the emitted light and amplify light emitted from the second aspherical lens.

Even if fluorescence by the same dye sample, that is, fluorescence of the same color, is generated by irradiating the excitation light having one wavelength to the various kinds of probes 240 in the multiplex PCR chips 200 to 700 And the probes 240, as shown in FIG. Therefore, it is possible to detect multiplex PCR products by using only one kind of light source and filter, without having to provide various light sources and filters. Likewise, even if the photodetector unit 1020 includes only one kind of filter, the multiplex PCR product can be detected. This configuration of the optical distributor 1010 and the optical detector 1020 can reduce the size of the optical equipment and reduce the equipment cost as well as reduce the time required for the detection as compared with the conventional multiplex PCR apparatus .

In addition, by monitoring in real time the reaction results by amplification of the nucleic acid in the reaction zone 224, particularly in the probe 240, during each cycle of the multiplex PCR in the multiplex PCR chips 200 to 700, The amplification and amplification degree of the nucleic acid sequence can be measured and analyzed in real time.

6A and 6B illustrate a multiplex PCR apparatus according to an embodiment of the present invention.

Referring to FIG. 6A, a multiplex PCR apparatus 1100 includes a substrate 1110; A first column block 900A disposed on the substrate 1110 and a second column block 900B spaced apart from the first column block 900A; And a driving unit 1130 for moving the chip holder 1120 and the chip holder 1120 on which the multiplex PCR chips 200 to 700 are mounted.

The substrate 1110 does not change its physical and / or chemical properties due to heating and temperature maintenance of the first column block 900A and the second column block 900B, RTI ID = 0.0 > 900B. ≪ / RTI > For example, the substrate 1110 may comprise or be made of a material such as plastic.

The first column block 900A and the second column block 900B are for maintaining the temperature for performing the denaturation step, the annealing step and the extension (or amplification) step for amplifying the nucleic acid. Gt; column block 900, and thus redundant description is omitted. Each of the thermal blocks 900A, 900B may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) step. For example, the thermal blocks 900A, 900B may maintain 50 占 폚 to 100 占 폚, preferably 90 占 폚 to 100 占 폚 when performing the denaturation step in the thermal blocks 900A, 900B, And may be maintained at 55 ° C to 75 ° C, preferably 72 ° C, when the annealing and extension (or amplification) steps are performed in the thermal blocks 900A and 900B. However, the present invention is not limited thereto, as long as it can perform the denaturation step, or the annealing and extension (or amplification) step. The first column block 900A and the second column block 900B may be spaced apart from each other by a predetermined distance such that mutual heat exchange does not occur. Thus, since heat exchange does not occur between the first column block 900A and the second column block 900B, in the nucleic acid amplification reaction which can be significantly affected even by a minute temperature change, the denaturation step, the annealing and the extension (Or amplification) stage. When the multiplex PCR chips 200 to 700 are contacted to one surface of each of the column blocks 900A and 900B, the first column block 900A and the second column block 900B are connected to the multiplex PCR chips 200, 700 can be heated and maintained at a temperature as a whole, so that the fluid in the multiplex PCR chips 200 to 700 can be uniformly heated and maintained at a temperature. Conventional multiplex PCR devices using a single column block have two column blocks, whereas the rate of temperature change in a single column block is in the range of 3 to 7 degrees Celsius per second, whereas the multiplex PCR device 1100 comprises two column blocks, The temperature change rate in the column blocks 900A and 900B of the second embodiment is within a range of 20 to 40 DEG C per second, thereby greatly shortening the multiplex PCR reaction time.

The chip holder 1120 may be equipped with the multiplex PCR chips 200 to 700. The inner wall of the chip holder 1120 may have a shape and a structure to be fixedly mounted on the outer wall of the multiplex PCR chips 200 to 700 so that the multiplex PCR chips 200 to 700 do not separate from the chip holder 1120 . In addition, the multiplex PCR chips 200 to 700 may be removable from the chip holder 1120. The chip holder 1120 may be operably connected to the driver 1130.

The driving unit 1130 can move the chip holder 1120 left and right and / or up and down on the thermal blocks 900A and 900B. Specifically, the driving unit 1130 may include all means for making the chip holder 1120 movable left and right and / or up and down over the first column block 900A and the second column block 900B. The chip holder 1120 can reciprocate between the first column block 900A and the second column block 900B by the right and left movement of the driving part 1130. By the up and down movement of the driving part 1130, The holder 1120 may contact and separate from the first column block 900A and the second column block 900B. The driving unit 1130 includes a rail 1132 extending in the left and right direction and a connecting member 1134 slidable in the left and right direction through the rail 1132 and slidable in the vertical direction, The chip holder 1120 may be disposed at one end of the connecting member 1134.

6B, the driving unit 1130 includes a multiplexer PCR chip 200 to 700 mounted on the chip holder 1120 and a PCR unit Reaction can be carried out.

First, the first column block 900A may be heated and maintained at a temperature for the denaturation step, e.g., 90-100 占 폚, and preferably heated and maintained at 95 占 폚. Also, the second column block 900B may be heated and maintained at temperatures for annealing and extending (or amplifying) steps, e.g., 55 占 폚 to 75 占 폚, and preferably heated and maintained at 72 占 폚.

The multiplex PCR chips 200 to 700 are moved downward by controlling the connection member 1134 of the driving unit 1130 after the multiplex PCR chips 200 to 700 are mounted on the chip holder 1120 or simultaneously, The first denaturation step of the multiplex PCR can be performed by contacting the chip holder 1120 equipped with the flex PCR chips 200 to 700 to the first column block 900A (step x).

Subsequently, the multiplexer PCR chips 200 to 700 are moved upward by controlling the connecting member 1134 of the driving unit 1130 so that the chip holder 1120 on which the multiplex PCR chips 200 to 700 are mounted is inserted into the first The first denaturation step of the multiplex PCR is terminated and the multiplex PCR chips 200 to 700 are moved on the second column block 900B through the rail 1132 of the driving unit 1130 (Step y).

Subsequently, the multiplexer PCR chips 200 to 700 are moved down by controlling the connecting member 1134 of the driving unit 1130 so that the chip holder 1120 on which the multiplex PCR chips 200 to 700 are mounted is moved to the second The first annealing and extension (or amplification) steps of multiplex PCR can be performed by contacting column block 900B (step z).

Lastly, the multiplexer PCR chips 200 to 700 are moved upward by controlling the connecting member 1134 of the driving unit 1130 so that the chip holders 1120, on which the multiplex PCR chips 200 to 700 are mounted, (Or amplification) step of the multiplex PCR is terminated and the multiplex PCR chips 200 to 700 are transferred through the rail 1132 of the driving part 1130 to the first column The nucleic acid amplification reaction can be performed by repeating the steps x, y and z after moving up the block 900A (circulation step).

Figure 7 shows a multiplex PCR device according to one embodiment of the present invention.

7, in the multiplex PCR apparatus 1200, the optical data providing unit 1010 and the optical detecting unit 1020 can be disposed with the first column block 900A and the second column block 900B therebetween have. A penetration portion 1136 for allowing light emitted from the light supplier 1010 to pass therethrough may be formed in the driving unit 1130 for optical measurement and the multiplex PCR chips 200 to 700 may be formed of a light transmitting material, It may be a light-transmitting plastic material.

7, the nucleic acid is amplified in the multiplex PCR chips 200 to 700 during the nucleic acid amplification reaction by the multiplex PCR device 1200 by the arrangement of the optical coupler 1010 and the optical detector 1020 Can be detected in real time. Specifically, the multiplex PCR chip makes a round trip between the first column block 900A and the second column block 900B to perform each step of the PCR reaction. In this process, the driving unit 1130 can stop the multiplex PCR chips 200 to 700 on the spaced space between the first column block 900A and the second column block 900B. At this time, light is emitted from the light providing part 1010, and the emitted light passes through the reaction area 224 of the multiplex PCR chips 200 to 700, specifically, the multiplex PCR chips 200 to 700 or the probe 240, The optical detecting unit 1020 can detect the optical signal generated by the amplification of the nucleic acid.

Thus, according to the multiplex PCR apparatus 1100, the reaction result by the amplification of the nucleic acid in the reaction region 224, particularly the probe 240, during each circulation step of the multiplex PCR reaction is monitored in real time The amount of the target nucleic acid sequence can be measured and analyzed in real time. 6A, 6B and 7 illustrate a multiplex PCR apparatus for performing a PCR reaction using two column blocks 900A and 900B. However, the multiplex PCR apparatus shown in FIG. The number of blocks may vary. For example, only one column block can be used for one multiplex PCR chip 200-700.

<Experimental Example>

1. Chip manufacturing process

A reaction probe and a probe coupling portion to be attached to a reaction region inside the chip were prepared. 5% to 40% of polyethylene glycol diacrylate (PEGDA), 5% to 40% of polyethylene glycol (PEG), 2-hydroxy-2-methyl- 1 to 10% of 2-Hydroxy-2-methyl-1-phenyl-1-propanone was prepared by adding TE buffer to prepare a prepolymer solution. And 0.1% to 30% of Tween-20 in a concentration ratio of 90% and 10% of the single strand DNA in which the prepolymer solution and the acridite and the PEG linker were combined. (SEQ ID NO: 1: Probe sequence: ACAGATGCCTTAACCTTTCCATGAGCGG). Thereafter, a multiplex PCR chip structure as shown in FIG. 2 was prepared, and a reaction probe and a probe-binding-site complex were attached to a porous structure formed in a reaction region inside the chip. Polyethylene glycol was removed using a washing buffer, Respectively. The finally prepared PCR chip is shown in Fig.

2. PCR reagent composition

The composition of the reagents was as follows: Positive control 1 (PC 1) and positive control 2 (PC 2) containing only probe in the porous structure of the PCR chip, Positive control 3 (PC 3) in which positive and reverse primers were added to positive control 1 and 2 (SEQ ID NO: 2: Forward primer sequence TGGTCATGGTGATGTTGATTACTATTCAG, SEQ ID NO: 3: Reverse primer sequence ACGTCTTACTTGCACTGATTGATTCA). The composition of each reagent is shown in Table 1 below.

PCR reagent composition Reagent composition Positive control group
(Gel: Probe only) Positive control group
(Gel: Probe only) Positive control group
(Gel: Primer / Probe) NBS Taqman
2X master mix 10 μl 10 μl 10 μl Primer 2 μl 2 μl - Template
(Target DNA) 1 μl 1 μl 1 μl DW 7 μl 7 μl 9 μl Total 20 쨉 l 20 쨉 l 20 쨉 l

3. Conditions for performing PCR

PCR conditions were pre-denaturation at 95 ° C for 8 seconds, denaturation at 95 ° C for 3 seconds, and annealing at 68 ° C, 14 ° C for a Taget gene sequence The annealing step was performed for 40 cycles (SEQ ID NO: 4: Target gene sequence: TAA TGA CCC TAA AGG TTT TAA CCT GAA GTA CCG TTA TGA ACT CGA TGA TAA CTG GGG AGT AAT AGG TTC GTT TGC TTA TAC TCA TCA GGG ATA TGA TTT CTT CTA TGG CAG TAA TAA GTT TGG TCA TGG TGA TGT TGA TTA CTA TTC AGT AAC AAT GGG GCC ATC TTT CCG CAT CAA CGA ATA TGT TAG CCT TTA TGG ATT ACT GGG GGC CGC TCA TGG AAA GGT TAA GGC ATC TGT ATT TGA TGA ATC AAT CAG TGC AAG TAA GAC GTC AAT GGC ATA CGG GGC AGG GGT GCA ATT CAA CCC ACT TCC AAA TTT TGT CAT TGA CGC TTC ATA TGA ATA CTC CAA ACT CGA TAG CAT AAA AGT TGG CAC CTG GAT GCT TGG TGC AGG GTA TCG ATT CTAA).

4. Results of PCR

PCR results were confirmed by three methods. FIG. 9 is an electrophoresis image of the positive control group 1, the positive control group 2, and the positive control group 3 from the left marker to the right. As a result, it can be confirmed that PCR was successfully performed inside the chip. FIG. 10 is a fluorescence image of a chip for positive control group 1, positive control group 2 and positive control group 3 from the left. According to this, according to this, PCR was successfully carried out through 40 circulation, and fluorescence expression was successfully performed specifically for the third porous structure . Figure 11 is a graph of fluorescence measurements for positive control (PC 1), positive control 2 (PC 2), and positive control 3 (PC 3), with positive control 1 (PC 1), positive control 2 PCR progress was confirmed in positive control group 3 (PC 3). Based on these results, it can be seen that the porous structure-based multiplex PCR apparatus according to the embodiment of the present invention can perform real-time PCR that can perform PCR more quickly than before, .

As described above, an optimal embodiment has been disclosed in the drawings and specification. Although specific terms have been employed herein, they are used for purposes of illustration only and are not intended to limit the scope of the invention as defined in the claims or the claims. Therefore, those skilled in the art will appreciate that various modifications and equivalent embodiments are possible without departing from the scope of the present invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> NANOBIOSYS Inc. <120> MULTIPLEX PCR CHIP AND MULTIPLEX PCR DEVICE COMPRISING THE SAME <130> WPN15020 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> aritificial probe sequence <400> 1 acagatgcct taacctttcc atgagcgg 28 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> aritificial primer sequence <400> 2 tggtcatggt gatgttgatt actattcag 29 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> aritificial primer sequence <400> 3 acgtcttact tgcactgatt gattca 26 <210> 4 <211> 400 <212> DNA <213> Artificial Sequence <220> <223> aritificial target gene sequence <400> 4 taatgaccct aaaggtttta acctgaagta ccgttatgaa ctcgatgata actggggagt 60 aataggttcg tttgcttata ctcatcaggg atatgatttc ttctatggca gtaataagtt 120 tggtcatggt gatgttgatt actattcagt aacaatgggg ccatctttcc gcatcaacga 180 atatgttagc ctttatggat tactgggggc cgctcatgga aaggttaagg catctgtatt 240 tgatgaatca atcagtgcaa gtaagacgtc aatggcatac ggggcagggg tgcaattcaa 300 cccacttcca aattttgtca ttgacgcttc atatgaatac tccaaactcg atagcataaa 360 agttggcacc tggatgcttg gtgcagggta tcgattctaa 400

Claims (9)

As a Multiplex Polymerase Chain Reaction (PCR) chip,
A plurality of probes for hybridization reaction that are specifically hybridized with different sequences of the nucleic acid molecules so as to simultaneously detect a plurality of nucleic acid molecules that are different from each other and are spaced apart from each other; And
A plurality of probe coupling parts disposed on an inner surface of the multiplex PCR chip to form a pore structure so as to increase a contact area between the probe and the nucleic acid molecule so that the probes are respectively coupled to the porous structure; &Lt; / RTI &
Wherein the probe comprises a fluorescent substance and a fluorescence inhibiting substance bound to the end or middle of the base sequence, respectively. The probe according to claim 1, wherein the probe is specifically hybridized with the sequence of the nucleic acid molecule, the fluorescence of the fluorescent substance is inhibited by the fluorescence inhibiting substance, and the probe is degraded Wherein the fluorescent material is released from the probe or is separated from the probe to generate fluorescence. [2] The multiplex PCR chip of claim 1, wherein the probe binding unit further comprises a plurality of primers that are specifically hybridized with different sequences of the nucleic acid molecules. The porous structure according to claim 1, wherein the porous structure is selected from the group consisting of polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDMA), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol diacrylate (EGDA), ethylene glycol dimethacrylate (EGDMA), polyvinyl alcohol (PVA), agarose, silicone, A porogen formed of at least one of a gel material composed of at least one of polyglycolic acid (PEG) and paraffin, polyethylene glycol (PEG) and ethylene glycol (EG) Wherein the primer is formed by adding the primer to the primer. The multiplex PCR chip according to claim 1, wherein the probe coupling unit comprises a linker material and a porous structure-binding material that bind to the fluorescent material. [4] The multiplex PCR chip of claim 3, wherein the probe coupling unit comprises a linker material and a porous structure-binding material that bind to the primer. The multiplex PCR chip of claim 1, wherein the multiplex PCR chip comprises: a first plate having a flat plate shape; A second plate disposed on the first plate and including an inlet, a reaction zone and an outlet; And a third plate disposed on the second plate to cover the reaction area and spaced apart from the lower surface of the probe, wherein the probe includes a central portion in which the probe is disposed, And a probe fixing section composed of a peripheral portion formed on the substrate. 8. A multiplex PCR chip according to any one of claims 1 to 7,
A light supplier for irradiating an excitation light toward the probe in the multiplex PCR chip; And
And an optical detector for detecting an emission light generated in the plurality of probes by the excitation light,
Wherein the optical detector and the detection by the optical detector are performed using light of a single wavelength or a plurality of wavelengths. 8. A multiplex PCR chip according to any one of claims 1 to 7, And
And at least one column block for contacting the multiplex PCR chip and transferring heat for multiplex PCR to the multiplex PCR chip.

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