KR20190095080A - Real-time polymerase Chain Reaction Apparatus for multiple diagnosis with single sample - Google Patents

Abstract

The present invention relates to a real-time PCR apparatus. More specifically, the present invention relates to the real-time PCR apparatus using a single sample which distributes the single sample to a plurality of reagent chambers and mixing channels formed on a PCR chip in the real-time PCR apparatus using a micro PCR chip, however, one sample chamber and a plurality of reagent chambers are sealed by using a plurality of mixing channels, and a surface of a sealing film closing a lower ends thereof is treated to have hydrophilicity to make samples injected into one sample chamber are simultaneously injected into multiple reagent chambers through multiple mixing channels, so that there is little time difference between samples injected into each reagent chamber. In addition, by distributing a sample through a number of closed mixing channels, a structure of an apparatus can be simplified by transferring the sample without force, and thus reliability for the single sample is improved, thereby being capable of multiple diagnosis by analyzing images of fluorescence emitted from a plurality of mixed channels after a PCR reaction.

Description

Real-time polymerase chain Reaction Apparatus for multiple diagnosis with single sample}

The present invention relates to a real-time PCR device, and more particularly, in a real-time PCR device using a micro PCR chip, a single sample is distributed to a plurality of chambers and channels formed in the PCR chip, and the fluorescence emitted from the plurality of channels after the PCR reaction. It is to provide a real-time PCR device capable of multi-diagnosis using a single sample to analyze the image to enable multi-diagnosis.

Today, the most commonly used method for detecting viruses and pathogens is a molecular diagnostic method that detects the causes and diseases of diseases by detecting nucleic acids (DNA and RNA or variants thereof) that cause diseases such as bacteria and viruses. . Molecular diagnostic method is composed of three steps, in order, pre-treatment to obtain pure DNA or RNA in the cell, gene amplification process using polymerase chain reaction (PCR) and the analysis process. That is, since the target gene can be amplified hundreds of millions through the PCR process, even a very small amount of the target substance can be analyzed with high sensitivity and specificity.

Therefore, amplification of target gene through PCR is essential for molecular diagnosis. PCR is an enzyme-based reaction that repeatedly amplifies a specific section of the base sequence of a target DNA to produce millions of strands of identical genetic material. Conventional PCR requires a relatively large amount of expensive reagents and a large thermal cycler, but miniaturized micro PCR chips require less reagent and have faster heat transfer, resulting in PCR times ranging from hours to tens of minutes. Can be greatly reduced. In addition, multiple element technologies can be integrated on the micro device and power consumption is low for driving the equipment. Miniaturized PCR chips can be developed as disposable chips to reduce cross-contamination or biochemical risk.

Polymerase Chain Reaction (PCR) repeats heating and cooling of genetic material including nucleic acids in sequence to replicate a portion having a specific sequence of the nucleic acid and exponentially extracts a nucleic acid having the specific sequence region. As a technology to amplify a nucleic acid, a specific area of nucleic acid can be amplified in a large amount in vitro, and in addition to the field of pure molecular biology, it can be applied not only to medicine, science, agriculture, veterinary medicine, food science, environmental science, but also archeology and anthropology. The range of application is expanding.

This PCR process consists of DNA denaturation, annealing and extension steps. DNA denaturation (Denaturation) is heat-treated at 94 ℃ for 15-30 seconds to separate the two strands of DNA into one strand of DNA, respectively. In the binding step, when the temperature is lowered after the heat treatment, two kinds of primers are annealed to the complementary part of a single stranded DNA. The most suitable temperature and time for annealing are determined by the base sequence and length of the primer, but they are usually annealed at 55 ° C for 30 seconds to 1 minute. In the extension step, DNA polymerase is applied to extend primers in the presence of four types of substrates (dNTPs). The time required for the extension reaction depends on the concentration of template DNA, the size of the amplified fragment, and the reaction temperature, but in general, the Thermus aquaticus (Taq) Polymerase is treated for 30 seconds to 10 minutes (amplification size of about 10 kbp) at 72 ° C. do. The time is set in consideration of the rate of DNA synthesis by Taq Polymerase (about 60 nucleotides / sec, 70 ° C).

After the PCR process, the amount of amplification of the target product is analyzed. For example, a sample of the reaction product is subjected to electrophoresis with an appropriate molecular weight marker using an agarose gel containing ethidium bromide. In this case, the formed band may be visualized by ultraviolet ray (transillumination). As described above, the amplification of the target gene can be confirmed using electrophoresis. However, the electrophoresis method only analyzes the amplification product after PCR to obtain information on the size of the amplified portion, and only speculates about the nucleotide sequence of the amplification site. It is possible. In addition, it takes about 2 to 3 days and has a disadvantage in that the stringency required for probe binding must be controlled under various conditions in order to be sure of specificity.

In order to compensate for the shortcomings of the electrophoresis method, a technique for analyzing the signal (fluorescence) from each cycle of the PCR to calculate the concentration of the amplification product present in the sample, and using a specific probe to obtain the PCR results quickly and accurately This so-called real-time PCR has been developed. Real-time PCR process is a PCR method which monitors the quantity of DNA synthesize | combined by a PCR reaction in real time using fluorescent substance.

In order to detect amplification products in real-time PCR in real time, i) using a nonspecific intercalator type such as SYBR Green inserted into any double-stranded DNA, or ii) having a complementary sequence for quantifying a target gene. There is a method using a sequence-specific DNA probe (probe type) consisting of oligonucleotides labeled with a fluorescent marker that can be detected only after binding of the probe (probe). The former can be used for multiple analysis, but can be used only if the variation of melting temperature (Tm) is large. The latter can be used for multiple detection of multiple target genes at the same time if different fluorescent dyes are used as probes.

In particular, the probe-type fluorescent material can be multi-detected for several target genes at the same time if different fluorescent dyes used as probes. However, in order to perform multiple detection using probe-type fluorescent materials, several probes labeled with fluorescent dyes having different wavelength bands should be used. As described above, when using multiple probes, interference between the probes is generated, which makes it difficult to design a probe for preventing the cost and to increase manufacturing costs. In addition, in order to distinguish and measure the fluorescence emitted from various fluorescent dyes, a plurality of light source modules and a plurality of light detection modules should be provided, which causes a problem in that the structure of the PCR device is complicated.

Meanwhile, a real-time PCR apparatus (thermal cycler) receives a PCR chip, a heating block for heating and cooling the PCR chip, a control unit for controlling the temperature of the heating block, a light source for irradiating light to the PCR chip, and receiving fluorescence emitted from the PCR chip. It includes a light detector for detecting. Therefore, the intensity of fluorescence emitted from a plurality of mixed channels provided in the PCR chip during the PCR process can be detected in real time to determine whether or not the amplification of the target gene.

However, in the case of using different types of fluorescent dyes (Reporter) for multiple detection, a plurality of light sources and a plurality of light detectors should be provided to distinguish and measure wavelengths emitted from various fluorescent dyes. As a result, the conventional real-time PCR apparatus has a problem that the structure of the apparatus is complicated and miniaturization is difficult for multiple detection.

Accordingly, it is possible to multi-detect, but by using a fluorescent material that emits fluorescence of the same wavelength band in response to the excitation light emitted from a single wavelength light source, the structure of the real-time PCR device can be simplified and reduced in size and weight. Development is being requested.

In addition, in order to perform multi-detection using a PCR chip, first, a sample containing a target gene needs to be equally divided into a plurality of mixing channels provided in the PCR chip. In the related art, each mixing channel is used by using a pipette or a dropper. Since a single sample is injected into the sample, different reagents may be placed on the sample or exposed to the outside to change its properties. Accordingly, there is a problem that the reliability of whether the sample dispensed in each mixing channel is a single sample.

Republic of Korea Patent Registration No. 10-1253455 (Registration Date: April 04, 2013)

The present invention is to solve the problems of the prior art, the main object of the present invention is to enable multi-diagnosis using a single sample to enable multiple diagnosis using a single sample in a real-time PCR device using a micro PCR chip. It is to provide a real-time PCR device.

In addition, when dispensing a single sample to a plurality of chambers and channels provided in the PCR chip for another purpose of the present invention to prevent contamination by injecting other reagents or foreign matter and to eliminate the time difference between the samples transferred to each chamber and the channel is sealed It is to provide a real-time PCR device capable of multi-diagnosis using a single sample that can improve the reliability of a single sample by transferring through the channel.

In addition, the present invention provides a real-time PCR device capable of multi-diagnosis using a single sample that can simplify the structure of the PCR chip by allowing the sample to be transferred to a plurality of chambers and channels with no power.

In addition, the present invention simplifies the structure, miniaturization and structure by RGB analysis of the image obtained by photographing the fluorescence emitted from a plurality of channels with a digital camera and determining the type and presence of the target gene based on this It is to provide a real-time PCR device capable of multiple diagnosis using a single sample that can be reduced in weight.

As a means for achieving the object of the present invention, a real-time PCR device capable of multiple diagnosis using a single sample according to the present invention,

A PCR chip comprising one sample chamber, a plurality of reagent chambers, a transparent substrate having a constant thickness, and a plurality of mixing channels connecting the sample chamber and the plurality of reagent chambers, and a sealing film attached to the bottom surface of the substrate;

A heating cooling module having a recess in which the PCR chip is seated to a predetermined depth;

A heating plate forming a bottom of the seating groove;

A thermoelectric element installed under the heating plate to heat or cool it;

A heat sink attached to a lower surface of the thermoelectric element and dissipating heat or cold air generated in the thermoelectric element;

A blower installed at a lower portion of the heat sink;

A light source provided at least of the PCR chip seated on the seating portion and irradiating light to a plurality of chambers or channels of the PCR chip;

A camera installed on the PCR chip to measure fluorescence emitted from each chamber or channel of the PCR chip;

A control unit installed inside the PCR device;

Is installed on the upper surface of the PCR device and electrically connected to the control unit includes a display unit for inputting or displaying information,

The camera acquires a fluorescence image by capturing fluorescence emitted by various kinds of reagents in response to the light irradiated from the light source, and the controller performs RGB analysis of the fluorescence image obtained by the camera and stores the result in a memory. It is characterized by diagnosing the target gene or disease in comparison with the information.

The light source includes a plurality of LEDs installed on one side of the PCR chip and corresponding to the plurality of reagent chambers to irradiate light toward the plurality of reagent chambers.

The light source is installed on both sides of the PCR chip and is installed to pass across the plurality of mixing channels on both sides of the plurality of mixing channels.

The plurality of LEDs installed in the light source emit light of a single wavelength band or light of different wavelength bands.

A plurality of LEDs installed in the light source irradiates light of different wavelength bands to react with the fluorescent dyes injected into the corresponding reagent chambers.

The heating cooling module is slidably installed back and forth, and the rear end of the seating recess is installed to secure the PCR chip seated in the seating groove by rotating up and down in accordance with the forward and backward of the heating cooling module.

 In the present invention, the PCR chip, one sample chamber is formed so as to pass through the substrate on one side of the substrate is formed with a sample inlet on the top; A plurality of reagent chambers formed at a predetermined depth on the other lower surface of the substrate; A plurality of mixing channels formed at a predetermined depth on the lower surface of the substrate and connecting the one sample chamber and the plurality of reagent chambers; A reagent inlet formed at a predetermined depth on an upper surface of the substrate so as to communicate with the reagent chamber; And a sealing film attached to a lower surface of the substrate to seal one sample chamber, a plurality of reagent chambers, and an open lower portion of the mixing channel, wherein the reagents are respectively injected into the plurality of reagent chambers through a reagent inlet. A single sample is injected into the sample chamber of the sample inlet, and the sealing film is coated to have a hydrophilicity to inject a single sample into the sample chamber while simultaneously transferring a single sample to a plurality of reagent chambers through a plurality of mixing channels without power. It is characterized in that the reliability of the single sample is improved.

The mixing channel may include a mixing channel portion having a diameter similar to the diameter of the reagent chamber and a narrow channel portion having a diameter smaller than the diameter of the mixing channel portion, wherein the mixing channel portion is formed to have a predetermined length on the reagent chamber. A channel portion is formed at a predetermined length on the sample chamber side and connected to the mixing channel portion, and a step is formed between the mixing channel portion and the narrow channel portion.

The surface of the sealing film may be hydrophilically treated to have hydrophilicity.

The sealing film may be treated such that the surface of the sealing film that closes the open lower surface of one sample chamber, a plurality of reagent chambers, and a plurality of mixing channels is partially hydrophilic.

The open top of the reagent inlet and the open top of the sample chamber are closed using a sticker attached to the top surface of the substrate.

The reagent injected into the reagent chamber is fixed to the bottom (surface of the sealing film) of the reagent chamber.

As described above, the multi-diagnosis real-time polymerase chain reaction apparatus according to the present invention is capable of multi-diagnosis using a single sample, it is possible to quickly distribute the sample without a separate device quickly and conveniently in the field Molecular diagnostics are effective.

In addition, the present invention by omitting a photodetector of a complex structure that can determine the target gene or diagnose a disease by analyzing the fluorescence image using a digital camera, the structure of the PCR device is simple and miniaturized to use or carry in the field It is convenient.

In addition, in the PCR chip according to the present invention, since one sample chamber is connected to a plurality of reagent chambers through a plurality of mixing channels, a sample may be rapidly dispensed into a plurality of mixing channels and reagent chambers in one sample injection. Since parallax rarely occurs between the reagents transferred to each reagent chamber and is not exposed to the outside air, reliability of a single sample is improved, and thus multiple diagnosis is possible.

In addition, the present invention is to treat the sample chamber and the plurality of reagent chambers and the mixing channel to the surface of the sealing vinyl sealing the open lower end to have a hydrophilic part of the plurality of mixing channels of the sample injected into the sample chamber And can be transferred quickly to the reagent chamber.

1 is a perspective view showing an example of a real-time PCR device capable of multiple diagnosis using a single sample according to the present invention,
2 is a perspective view showing an example of a PCR chip according to the present invention;
2 is a cross-sectional view showing the structure of a heating cooling module according to the present invention shown in FIG.
3 is a plan view showing an example of a light source installed in the heating cooling module shown in FIG.
4 is a perspective view of a PCR chip according to the present invention,
5 is a plan view of a PCR chip according to the present invention shown in Figure 4,
6 is a cross-sectional view of a PCR chip according to the present invention shown in Figure 4,
7 and 8 are cross-sectional views showing the operation of the real-time PCR device capable of multiple diagnosis using a single sample according to the present invention shown in FIG.
9 is a plan view showing another embodiment of a light source according to the present invention;
10 and 11 are a side view and a plan view showing a process of dispensing a sample using a PCR chip according to the present invention.

Advantages and features of the present invention, and a method for achieving the same will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. However, this embodiment is provided to explain in detail enough to easily implement the technical idea of the present invention to those skilled in the art. In addition, in describing the embodiments of the present invention, when it is determined that the detailed description of the related well-known configuration or function interferes with the understanding of the embodiments of the present invention, the detailed description thereof will be omitted.

-Definition of Terms-

First, terms used in the present patent specification are defined.

As used herein, the term "polymerase chain reaction" or "polymerase chain reaction" refers to a reaction that amplifies a particular target nucleic acid molecule using a heat stable DNA polymerase. In addition to DNA polymerase, PCR includes reactions containing ^bivalent ions such as primers (forward primers and reverse primers), deoxynucleotide triphosphate mixtures, and Mg ²⁺ , which are oligonucleotides that can hybridize specifically to target nucleic acids. Mixtures are used. DNA molecules produced by such PCR reactions are referred to herein as "amplification products".

As used herein, the term "primer" is used to initiate a PCR reaction, meaning oligonucleotides or polynucleotides that hybridize complementarily to template DNA. A primer for a PCR reaction is a pair of forward primers (or sense primers) selected from the same sense strand as the direction of genetic code of the nucleic acid molecule to be amplified and reverse primers (or antisense primers) selected from antisense strands complementary to the sense strands. Used.

As used herein, the term "real-time PCR device" can amplify a nucleic acid having a specific base sequence, and can measure and analyze the occurrence and extent of PCR products in real time. In addition, 'multi-diagnosis' is to identify or analyze various diseases at once by performing a multiplex polymerase chain reaction (PCR) to amplify various nucleic acids using a single sample.

As used in this patent specification, the term 'PCR process' means that a sample solution containing double-stranded DNA is heated to a specific temperature, for example about 95 ° C., in order to amplify deoxyribonucleic acid (DNA) having a specific nucleotide sequence. DNA denaturing step of separating helical DNA into single stranded DNA, providing oligonucleotide primers having sequences complementary to the specific sequencing to be amplified in the sample solution, An annealing step in which the primer is bound to a specific nucleotide sequence of the single stranded DNA by cooling to a temperature, for example, 55 ° C. to form a partial DNA-primer complex, and the sample solution is subjected to an appropriate temperature, for example, after the annealing step. For example, the DNA is maintained at 72 ℃ to form a double-stranded DNA based on the primer of the partial DNA-primer complex by DNA polymerase Performing the steps St. (extension step), and a step 3 for example, it is possible to amplify a DNA having a specific base sequence is repeated 20 to 40 times the circuit exponentially. In this case, in some cases, the PCR device may simultaneously perform the annealing step and the synthesis step.

As used herein, the term 'sample' refers to the genetic material to be amplified or a biological solution containing such genetic material. 'Reagent' includes fluorescent dyes, probes, and the like. And the 'master mixture' refers to a mixture containing a primer (Trimer), DNA polymerase (Taq polymerase or DNA polymerase), dNTP, buffer (buffer solution), magnesium chloride (MgCl ₂ ). Here, as a genetic material to be used as a template, plasmid DNA, genomic DNA, cDNA, total RNA, etc. are usually used, and the primer consists of a pair of primers of 25 to 40 bp in length capable of binding to both ends of a specific region of the target gene. In addition, DNA polymerase uses an enzyme which does not lose activity even at a high temperature of more than 90 ℃. dNTP is a nucleotide that is a synthetic material and consists of dATP, dCTP, dGTP, and dTTP. In addition, Mg ²⁺ is required for the DNA polymerase to function and for the binding of dNTP and primer. Buffers are necessary for the activity of the polymerase.

Hereinafter, with reference to the accompanying drawings will be described in detail a preferred embodiment of a real-time PCR device (polymerase chain reaction device) capable of multiple diagnosis using a single sample according to the present invention.

1 is a perspective view showing an example of a real-time PCR device (1) capable of multiple diagnosis using a single sample according to the present invention, Figure 2 is a cross-sectional view showing the structure of a heating cooling module according to the present invention shown in FIG. 3 is a plan view illustrating an example of a light source installed in the heating cooling module illustrated in FIG. 2.

As shown, the real-time PCR device 1 (hereinafter referred to as 'polymerase chain reaction device or PCR') capable of multiple diagnosis using a single sample of the present invention is largely the PCR device main body 2 and the above. It includes a PCR chip (5) made to be inserted into the PCR device body (2).

 The front surface of the main body case 4 is provided with a heating and cooling module 7 which is slidably installed in the front and rear through the opening 41. The selection knob 8 and the display panel 9 are provided on the upper surface of the main body of the PCR device 2.

The PCR chip 5 is for amplifying a large amount of target genes through a cooling heating cycle, and is attached to a transparent substrate 51 having a predetermined thickness and a lower surface of the substrate 51 having a plurality of chambers and a mixing channel. Film 52.

Specifically, the heating and cooling module 7 is formed in a rectangular block shape to be inserted into the body case 4 through the opening 41. In addition, a mounting groove 71 is formed at a predetermined depth so that the PCR chip 5 may be seated on an upper surface thereof. In addition, the rear end of the seating groove 71 is rotated up and down in accordance with the forward and backward of the heating and cooling module (7) for fixing the PCR chip (5) seated in the seating groove 71 Is installed.

2 is a cross-sectional view showing an example of the heating cooling module 7 according to the present invention. As shown, the heating cooling module 7 includes a heating unit 73, a blowing fan 74 and a light source 75. The heating part 73 is for heating and cooling the PRC chip 5 seated in the seating recess 71, a heating plate 731 forming a bottom of the seating recess 71, and the heating plate ( The thermoelectric element 732 is installed in the lower portion of the 731 and heats or cools it, and is attached to the lower surface of the thermoelectric element 732 and wind is blown by the blower fan 74 in the thermoelectric element 732. And a heat sink 734 which releases generated heat or cold air.

The heating plate 731 is made of a material having excellent heat transfer rate, and is made of a flat plate so as to be in close contact with the bottom surface of the PCR chip 5. The thermoelectric element 732 is heated or cooled according to the direction of the current applied from the outside. Hot and cold air generated in the thermoelectric element 732 is transferred to the PCR chip 5 through the heating plate 731. At this time, since the sealing film 52 attached to the lower surface of the PCR chip 5 is made of a thin thin film, heat or cold of the heating plate 731 is quickly transmitted. The heat dissipation plate 734 is made of a material having excellent thermal conductivity, and a plurality of heat dissipation fins are formed to protrude downward, and heat or cold air generated from the thermoelectric element 732 using wind blown by the blower fan 74. By emitting the light to the outside, the efficiency of the thermoelectric element 732 is improved.

And a light source 75 is installed on one side of the seating groove (71). As shown in FIG. 6, the light source 75 includes a plurality of LEDs 751 installed to irradiate light to one side of the PCR chip 5 seated in the seating recess 71 and a printed circuit in which the light source 75 is installed. And a substrate 752. The plurality of LEDs 751 are formed to correspond to each other so as to irradiate light toward the plurality of reagent chambers 55. In addition, an auxiliary LED may be further installed between the plurality of LEDs 751 installed to correspond to the reagent chamber 55 to irradiate surface light.

Preferably, the plurality of LEDs 751 may irradiate light of a single wavelength band or irradiate light of different wavelength bands. Alternatively, some may irradiate light in a single wavelength band and others may irradiate light in different wavelength bands. In particular, in the case of irradiating light of different wavelength bands, multi-diagnosis is possible in consideration of the reaction characteristics of the fluorescent dye injected into the corresponding reagent chamber 55.

Subsequently, as illustrated in FIG. 4, the substrate 51 has a square plate shape made of a transparent polymer material. Two vertical walls 511 protrude from both sides of the upper surface of the substrate 51 so as to easily hold and move the substrate. One sample chamber 53 is formed at one side of the substrate 51 to penetrate the substrate 51. The sample chamber 53 penetrates up and down the substrate 51. And the open upper end of the sample chamber 53 serves as a sample inlet (53a) for injecting a sample. The open lower portion of the sample chamber 53 is closed by the sealing film 52.

Subsequently, a plurality of reagent chambers 55 are formed at a predetermined depth on the other lower surface of the substrate 51. Preferably, the reagent chamber 55 is circular and a plurality of reagent chambers 55 are arranged in a row. The reagent chamber 55 is formed at a constant depth on the lower surface of the substrate 51, and a reagent inlet 55a is formed at a predetermined depth on the upper surface of the reagent chamber 55 to inject the reagent into the reagent chamber 55. The diameter of the reagent inlet 55a is smaller than the diameter of the reagent chamber 55 such that a step 55b is formed between the reagent chamber 55 and the reagent inlet 55a. The opened lower portion of the reagent chamber 55 is closed by the sealing film 52 attached to the lower surface of the substrate 51.

In addition, a plurality of mixing channels 54 connecting one sample chamber 53 and a plurality of reagent chambers 55 are formed at a predetermined depth on the lower surface of the substrate 51. As shown in FIG. 6, the mixed channel 54 includes a mixed channel portion 541 and a narrow channel portion 542. The mixing channel portion 541 is formed to a predetermined length on the reagent chamber 55 side and the narrow channel portion 542 is formed to a predetermined length on the sample chamber 53 side and extends to meet the mixing channel portion 541. . In this case, the mixing channel portion 541 has a width and height similar to that of the reagent chamber 55, and the narrow channel portion 542 has a width and height smaller than that of the mixing channel portion 541. Therefore, a step 543 of a constant width and height is formed between the mixing channel portion 541 and the narrow channel portion 542.

Therefore, the sample supplied to the sample chamber 53 may move to the reagent chamber 55 through the narrow channel portion 542 and the mixing channel portion 541. However, the reagent or mixture injected into the reagent chamber 55 does not flow back into the sample chamber 53 due to the narrow channel portion 542. That is, the narrow channel portion 542 prevents the reagent or mixture from flowing back from the reagent chamber 55 to the sample chamber 53. In addition, the open upper end of the reagent inlet 55a and the open upper end of the sample inlet 53 may be sealed by a sticker 57 attached to the upper surface of the substrate 51.

On the other hand, the sealing film 52 serves to close the opened lower portions of the plurality of reagent chambers 55, the mixing channel 54 and one sample chamber 53 attached to the lower surface of the substrate 51. In addition, the sealing film 52 is made of a thin film to be able to quickly transfer the heat or cold air of the heater unit installed below. To this end, the sealing film 52 may be made of a metal having excellent heat transfer efficiency or may be made of a polymer material including nano carbon or carbon tube.

In addition, the sealing film 52 also serves to quickly transfer the sample injected into the sample chamber 53 to the plurality of reagent chambers 55 through the plurality of mixing channels 54 with no power. To this end, the surface of the sealing film 52 should be treated to have hydrophilicity. That is, since the sealing film made of ordinary plastic is hydrophobic or positively charged on its surface, it is not quickly conveyed or is unevenly distributed when a sample having a charge is injected into the sample chamber 53. There is. In order to solve this problem, conventionally, the substrate 51 is tilted inclined or the sample is transferred using a power such as a pump or centrifugal force, but there is a problem in that the structure becomes complicated. However, in the present invention, the sealing film 52 is treated such that the surface has hydrophilicity by chemical treatment, ultraviolet irradiation, plasma treatment, etc., so that the sample can be transported without force.

On the other hand, the sealing film 52 can be treated to have hydrophilicity by using a vacuum plasma treatment, but the effect of hydrophilicity does not last long when exposed to air. In addition, the hydrophilic sealing film 52 has a problem that the sealing film 52 cannot be attached to the lower surface of the substrate 51 because the adhesive does not adhere well. Accordingly, the sealing film 52 partially hydrophilizes the surface of the sealing film that closes the open lower surface of one sample chamber, a plurality of reagent chambers, and a plurality of mixing channels. For example, in the PCR chip 5 of the present invention, the sealing film 52 is adhered to the lower surface of the substrate 51 and then subjected to vacuum plasma treatment. The surface of the sealing film 52 attached to the lower surface of the substrate 51 is hydrophilically treated through the sample inlet 53a and the plurality of reagent inlets 55a of the sample chamber 53. That is, only the surface of the sealing film closing the sample chamber 53, the plurality of reagent chambers 55, and the plurality of mixing channels 54 may be hydrophilically treated. And the vacuum plasma-treated PCR chip (5) is kept in a gas-tight bag made of aluminum so as not to contact with the outside air so that the hydrophilic effect can be maintained. However, of course, the substrate 51 or the sealing film 52 as a whole can also be water-soluble.

A protrusion 58 is formed on the lower surface of the substrate 51 at a predetermined height along the sample chamber 53, the plurality of reagent chambers 55, and the mixing channel 54. In addition, protrusions 59 are also formed in a quadrangular shape so as to surround them. These protrusions 58 and 59 minimize the contact area between the sealing film 52 and the substrate 51 to facilitate the adhesion of the sealing film 52. In addition, the upper surface of the substrate 51 may be provided with a sticker 57 for closing the sample inlet (53a) and a plurality of reagent inlet (55a).

As such, the microchip 5 may rapidly dispense a single sample injected into one sample chamber 53 into a plurality of reagent chambers 55 through a hydrophilic surface of the sealing film 52. . In addition, since the sample is dispensed through the sealed mixing channel 54, the sample is not contacted with the outside air without using a pipette or a tip in the process of dispensing the sample. In addition, a narrow channel portion 542 is formed in each mixing channel 54 to prevent the reagents or mixtures in each reagent chamber 55 from flowing back to the sample chamber 53, thereby preventing mixing of different reagents. Because no other reagents or foreign substances are introduced, the reliability of a single sample is increased.

Meanwhile, reagents are injected into the plurality of reagent chambers 55 through a reagent inlet 55a. For example, reagents include fluorescent dyes, probes, and the like. In addition, the reagent injected into the reagent chamber 55 may be lyophilized and fixed to the bottom of the reagent chamber 55, that is, the surface of the sealing film 52. In this case, since the operation of injecting different reagents into the plurality of reagent chambers 55 is required for precision, it may be performed by an expert in a laboratory. Therefore, in the field, it is possible to react different reagents and samples simply by injecting the collected sample into one sample chamber 53.

Preferably, a single sample is injected into the one sample chamber 53. Herein, a single sample refers to a biological sample containing a genetic material that is a template to be amplified. In addition, the sample may include a master mixture including a primer (Trimer), DNA polymerase (Taq polymerase or DNA polymerase), dNTP, buffer (buffer solution), magnesium chloride (MgCl ₂ ). The sample injected into the sample chamber 53 may be quickly transferred along the surface of the hydrophilic sealing film 52. That is, when the sample is injected into the sample chamber 53, the sample is distributed evenly into the plurality of reagent chambers 55 through the plurality of mixing channels 54 at the same time as the injection.

In addition, different reagents are injected into the reagent chamber 55. The reagent comprises a fluorescent dye, preferably a plurality of different reagent dye in the reagent chamber (55). Probes are included. Preferably, the reagents injected into the plurality of reagent chambers 55 may be fixed to the bottom of the reagent chamber 55, that is, the surface of the sealing film 52 through a separate freeze drying process. As described above, the operation of injecting various reagents into the plurality of reagent chambers 55 is performed in a laboratory, and in the field, the sample is injected into one sample chamber 53 and fixed to the plurality of reagent chambers 55. After dissolving, the PCR chip 5 may be placed in a PCR device to perform a PCR process.

Referring back to Figures 1 and 2, the upper surface of the heating and cooling module 7 is further provided with a support 32 for pressing and fixing the PCR chip (5) seated in the seating groove (71). The holder 32 is provided with a pressing plate 34 having a size and shape similar to that of the PCR chip 5, and the viewing plate 35 is formed on the pressing plate 34 with a constant size. The viewing window 35 is formed to correspond to the plurality of mixing channels 54 formed on the PCR substrate 5 so that the fluorescence emitted from the plurality of mixing channels 54 can be photographed using a camera to be described later. And the hinge 36 is installed at the lower end of the fixing base 32 to be coupled to the rear end of the mounting groove 71. At this time, the hinge 36 is provided with a restoring spring. Accordingly, when the heating cooling module 7 slides back and forth, the holder 32 is rotated back and forth in contact with the front surface of the body case (4). In addition, the rail 47 is installed on the side of the heating and cooling module 7 to slide smoothly.

Next, Figure 7 is a cross-sectional view showing the internal structure of the PCR device main body 2 according to the present invention. As shown, a camera 41 and a control unit 42 are installed in the inner space of the main body case 4, and the display panel 8 is rotatably installed up and down on the upper surface of the main body case 4. . In addition, a power supply unit 43 is installed inside the main body case 4.

The camera 41 photographs the fluorescence emitted from the fluorescent dye by reacting with the light irradiated from the light source 75. The camera 41 is installed on the upper portion of the camera 41 so as to photograph a plurality of mixed channels 54 at once. Preferably, the camera 41 uses an image sensor such as a CCD or a CMOS as a digital camera. In addition, various lenses and filters can be used. Accordingly, the camera 41 acquires an image by capturing fluorescence emitted from the plurality of mixing channels 54 at regular time intervals during the PCR process.

 In addition, the controller 42 is electrically connected to the thermoelectric element 732, the blower fan 74, the selection knob 8, the display panel 9, and the like to control the overall device and to the camera 41. And analyze the acquired image. To this end, the controller 42 includes a microprocessor and a memory. The microprocessor analyzes the color (RGB) of the image captured by the camera 41. To this end, the memory is equipped with an RGB analysis program. The microprocessor runs an RGB analysis program and analyzes the RGB of the fluorescence emitted from each mixed channel 34 and converts the RGB into a constant ratio. For example, 'RGB (255,0,0)' is determined as red because green and blue are 0. If it is RGB (255, 100, 0), it can be judged as light red mixed with green.

In the memory, various kinds of target genes or diseases are stored using a table according to the type (RBG ratio) and intensity of fluorescence color emitted in response to the wavelength range of the light source 75. Therefore, the microprocessor compares the color (RBG ratio) emitted from each reagent chamber 55 with the target gene or disease type stored in the table. The result is stored in memory and displayed on the display panel.

On the other hand, as shown in Figure 8, the inside of the main body case 4, the heating cooling module 7 is slidably installed back and forth. For example, when the heating cooling module 7 moves forward to be separated from the main body case 4, the power connector 431 formed at the rear end of the main body case 4 is separated to the heating cooling module 7. The power supply is cut off. On the contrary, when the heating cooling module 7 is moved backward and inserted into the main body case 4, the power connector 431 formed at the rear end of the main body case 4 is coupled to supply power to the heating cooling module 7. do. In addition, a switch is further installed on an upper surface of the heating cooling module 7. The switch is turned on or off in contact with the body case 4. For example, when the heating cooling module 7 moves forward, the switch is turned on, and when the rear cooling module 7 is retracted backward, the main body case 4 and the switch come into contact with the switch.

9 shows another embodiment of a light source installed in the heating cooling module 7, and light sources 75a and 75b are installed at both sides of the seating recess 71. The two light sources 75a and 75b are provided with a plurality of LEDs 751 installed to irradiate light across a plurality of mixing channels 54 of the PCR chip 5 seated in the seating recess 71. And a printed circuit board 752 in which they are installed. The plurality of LEDs 751 are correspondingly formed to cross the plurality of mixing channels 54. In addition, an auxiliary LED may be further installed between the plurality of LEDs 751 to irradiate surface light. Preferably, the plurality of LEDs 751 may irradiate light of a single wavelength band or irradiate light of different wavelength bands. Alternatively, some may irradiate light in a single wavelength band and others may irradiate light in different wavelength bands. In particular, in the case of irradiating light of different wavelengths, multiple diagnosis is possible in consideration of the reaction characteristics of the fluorescent dyes injected into the plurality of mixed channels 54.

Hereinafter, a method of quantitatively dispensing a single sample using a PCR chip according to the present invention.

10 is a cross-sectional view illustrating a process of quantitatively dispensing a single sample using a PCR chip according to the present invention, and FIG. 11 is a plan view illustrating a process of quantitatively dispensing a single sample using a PCR chip according to the present invention.

First, FIGS. 10A and 11A show an example of a PCR chip 5 according to the present invention. The PCR chip 5 of the present invention is supplied in a sealed state in an aluminum bag in the manufacturing step. As shown, it consists of one sample chamber 53 and a plurality of reagent chambers 55 and a plurality of mixing channels 54 connecting the sample chamber 53 and the plurality of reagent chambers 55.

10B and 10B show the injection of reagents (primers and PCR MIX) into the plurality of reagent chambers 55 using a pipette. Preferably, the step of injecting reagents into the reagent chamber 55 of various kinds of reagents may be performed by an expert in a laboratory or by using an automated device. Then, the PCR chip 5 into which the reagent is injected is put into lyophilization and freeze-dried to fix the reagent to the bottom of the reagent chamber 55. In this way, by fixing the reagent on the floor, it is possible to prevent the reagent from moving or leaking while transferring the PCR chip 5.

10C and 11C show a state in which a sample is injected into the sample chamber 53. The operation of injecting the sample into the sample chamber 53 is performed at the site where the sample is collected. When a sample is injected into the sample chamber 53 using a pipette, as shown in FIGS. 10D and 11D, the injected sample is immediately transferred to the plurality of reagent chambers 55 through the plurality of mixing channels 54. That is, the sample is quickly transferred to the plurality of reagent chambers 55 without force along the hydrophilic surface of the sealing film 52. The sample transferred to the reagent chamber 55 is mixed with the reagent by melting the fixed reagent. The mixed solution in which the reagent and the sample are mixed is filled in the plurality of reagent chambers 55 and the mixing channel 54. However, the mixed liquid in the mixed channel 54 does not flow back to the sample chamber 53 due to the narrow channel portion 642. Then, the sticker 57 is attached to the upper surface of the PCR chip 5 to seal the injection hole.

Subsequently, as shown in FIGS. 7 and 8, the PCR chip 5 in which a single sample is dispensed is seated in a seating groove 71 formed on an upper surface of the heating cooling block 7. When the heating and cooling block 7 is inserted into the main body case 4, the fixing base 32 presses and fixes the upper surface of the PCR chip 5. The heating unit 73 repeats a heating and cooling cycle to amplify target genes in the plurality of reagent chambers 55 and the mixing channel 54. In the PCR process, the fluorescent material binds to the gene and reacts with the light irradiated from the light source 75 to emit a predetermined fluorescence. Then, the camera 41 installed on the PCR chip 5 acquires an image by photographing fluorescence emitted from the plurality of mixing chambers 54. In this case, the camera 41 may measure fluorescence through the viewing window 35 formed on the fixing part 32. The see-through window 35 has a rectangular shape to block light emitted directly from the light source 75. Preferably, the viewing window 35 may be provided with a plurality of blocking walls partitioned to correspond between the plurality of mixing channels 34. The barrier wall prevents interference from being caused by fluorescence emitted from neighboring mixing chamber 34.

Subsequently, the controller 42 analyzes the digital image obtained from the camera 41 by an RGB analysis program and displays RGB at a constant numerical ratio. In addition, the ratio of the analyzed RGB is determined in consideration of the wavelength band of the light source and the type of reagent, and the presence or absence of the target gene and the disease by comparing with the RGB ratio expected from the target gene and the disease. The result can be displayed to the outside through the display panel 9 installed on the upper surface of the PCR apparatus main body 2.

As such, the present invention can be multi-diagnostic using a single sample. That is, in the present invention, different fluorescent dyes are injected into the plurality of reagent chambers 55 of the PCR chip 6, and then a single sample, that is, a sample containing a target gene and a master mix, is injected into each of the reagent chambers 55. Multi-diagnosis is possible by supplying the plurality of reagent chambers 55 and the mixing channel 54. At this time, the light source 75 allows the fluorescent material injected into the reagent chamber 55 to emit light of a wavelength band that can react.

In addition, in order to perform multiple diagnosis by injecting a single sample into the plurality of reagent chambers 53, it is necessary to ensure that the sample supplied to each reagent chamber 55 is a single sample. That is, when the samples supplied to the plurality of reagent chambers 55 are mixed with different reagents or foreign substances are mixed, the reliability of a single sample may be deteriorated. For example, conventionally a single sample has been injected into several reagent chambers while exchanging multiple pipettes or pipette tips. Then, a single sample is different because the time that the sample is injected into each reagent chamber 55 is different, and the reagent exposed to the outside air or the pipette may enter the other reagent chamber 55 during the injection of the sample into the plurality of reagent chambers 55. The reliability for

However, in the PCR chip 5 according to the present invention, since one sample chamber 53 is connected to a plurality of reagent chambers 55 through a plurality of mixing channels 54, a plurality of mixing channels (for example, one sample injection) 54) and the sample is dispensed into the reagent chamber 55, there is no fear of contamination by different reagents. In addition, when the sample injected into the sample chamber 53 is instantaneously transferred through the hydrophilic surface of the sealing film 52, the parallax between the samples supplied to each reagent chamber 55 hardly occurs and the mixed mixing channel 54 is closed. Because it is supplied through), there is little possibility of foreign substances from entering the outside air. Therefore, according to the PCR chip 5 of the present invention, the reliability of a single sample is improved, and multiple diagnosis is possible.

As described above, the real-time polymerase chain reaction apparatus 1 capable of multi-diagnosis according to the present invention may be multi-diagnosed using a single light source or multi-diagnostic using a light source of various wavelengths depending on the type of light source. In addition, a variety of diseases can be diagnosed using a single sample or various samples using various PCR chips of the present invention. In addition, the PCR chip 5 according to the present invention can distribute the sample evenly without a separate device, it is convenient to use in the field. In addition, since the present invention obtains a digital image using a digital camera and analyzes the color to determine a target gene or diagnoses a disease, the structure of the PCR apparatus main body is simple and small to omit a complex photodetector, It is convenient to carry.

 In the present specification and drawings have been described with respect to preferred embodiments of the present invention, although specific terms are used, it is only used in a general sense to easily explain the technical details of the present invention and help the understanding of the invention, the present invention It is not intended to limit the scope of. In addition, the present invention is not limited to the embodiments disclosed above, but may be implemented in various forms. The present embodiments are merely provided to complete the disclosure of the present invention, and to fully inform the scope of the invention to those skilled in the art, and the present invention is described only in the claims. It is only defined by the scope of the claims.

1: Real-time PCR device capable of multiple diagnosis using a single sample
2: PCR apparatus body 4: body case
5: PCR chip 7: heating cooling module
8: Select Knob 9: Display Panel
51: substrate 52: sealing film
53: sample chamber 54: mixing channel
55: reagent chamber 55: sample inlet
57: sticker 58,59: protrusion
541: mixed channel portion 542: narrow channel portion
543: step 32: holder
71: seating groove 73: heating unit
74: blower fan 75: light source
731: heating plate 732: thermoelectric element
734: heat sink

Claims (12)

A PCR chip comprising one sample chamber, a plurality of reagent chambers, a transparent substrate having a constant thickness, and a plurality of mixing channels connecting the sample chamber and the plurality of reagent chambers, and a sealing film attached to the bottom surface of the substrate;
A heating cooling module having a recess in which the PCR chip is seated to a predetermined depth;
A heating plate forming a bottom of the seating groove;
A thermoelectric element installed under the heating plate to heat or cool it;
A heat sink attached to a lower surface of the thermoelectric element and dissipating heat or cold air generated in the thermoelectric element;
A blower installed at a lower portion of the heat sink;
A light source provided at least of the PCR chip seated on the seating portion and irradiating light to a plurality of chambers or channels of the PCR chip;
A camera installed on the PCR chip to measure fluorescence emitted from each chamber or channel of the PCR chip;
A control unit installed inside the PCR device;
Is installed on the upper surface of the PCR device and electrically connected to the control unit includes a display unit for inputting or displaying information,
The camera acquires a fluorescence image by photographing fluorescence emitted by various kinds of reagents in response to light irradiated from the light source, and the control unit performs RGB analysis of the fluorescence image obtained by the camera and stores the result in memory. Real-time PCR device capable of multiple diagnosis using a single sample, characterized in that for diagnosing the target gene or disease in comparison with. According to claim 1,
The light source includes a plurality of LEDs installed on one side of the PCR chip and include a plurality of LEDs corresponding to the plurality of reagent chambers to irradiate light toward the plurality of reagent chambers. This real time PCR device available. According to claim 1,
The light source may be installed on both sides of the PCR chip and may include a plurality of LEDs installed to pass across the plurality of mixing channels on both sides of the plurality of mixing channels. Real time PCR device. The method according to claim 2 or 3,
A plurality of LEDs installed in the light source is a real-time PCR device capable of multi-diagnosis using a single sample, characterized in that irradiating light of a single wavelength band or light of different wavelength bands. The method according to claim 2 or 3,
A plurality of LEDs installed in the light source is a real-time PCR device capable of multi-diagnosis using a single sample, characterized in that for irradiating the light of different wavelengths to react with the fluorescent dye injected into each corresponding reagent chamber. According to claim 1,
The heating cooling module is slidably installed back and forth through the opening formed in the front of the PRC device, the rear end of the seating groove is rotated up and down in accordance with the forward and backward of the heating cooling module PCR chip seated in the seating groove Real-time PCR device capable of multi-diagnosis using a single sample, characterized in that the holder is installed to fix the According to claim 1,
The PCR chip may include: a sample chamber formed at one side of the substrate to penetrate the substrate and having a sample inlet formed thereon; A plurality of reagent chambers formed at a predetermined depth on the other lower surface of the substrate; A plurality of mixing channels formed at a predetermined depth on the lower surface of the substrate and connecting the one sample chamber and the plurality of reagent chambers; A reagent inlet formed at a predetermined depth on an upper surface of the substrate so as to communicate with the reagent chamber; And a sealing film attached to a lower surface of the substrate to seal one sample chamber, a plurality of reagent chambers, and an open lower portion of the mixing channel, wherein the reagents are respectively injected into the plurality of reagent chambers through a reagent inlet. A single sample is injected into the sample chamber of the sample inlet, and the sealing film is coated to have a hydrophilicity to inject a single sample into the sample chamber while simultaneously transferring a single sample to a plurality of reagent chambers through a plurality of mixing channels without power. Real-time PCR device capable of multi-diagnosis using a single sample, characterized in that the reliability of the single sample is improved. The method of claim 7, wherein
The mixing channel may include a mixing channel portion having a diameter similar to the diameter of the reagent chamber and a narrow channel portion having a diameter smaller than the diameter of the mixing channel portion, wherein the mixing channel portion is formed to have a predetermined length on the reagent chamber. The channel portion is formed in a predetermined length on the sample chamber side is connected to the mixed channel portion, the real-time PCR device capable of multiple diagnosis using a single sample, characterized in that the step is formed between the mixed channel portion and the narrow channel portion. According to claim 1,
The surface of the sealing film is a real-time PCR device capable of multi-diagnosis using a single sample, characterized in that the hydrophilic treatment to have a hydrophilic treatment. The method of claim 9,
The sealing film is a real-time PCR capable of multi-diagnosis using a single sample, characterized in that the surface of the sealing film to close the open lower surface of one sample chamber, a plurality of reagent chambers and a plurality of mixing channels is partially hydrophilic Device. The method of claim 7, wherein
The open top of the reagent inlet and the open top of the sample chamber is closed using a sticker attached to the upper surface of the substrate real-time PCR device capable of multiple diagnosis using a single sample. The method of claim 7, wherein
The reagent injected into the reagent chamber is a real-time PCR device capable of multi-diagnosis using a single sample, characterized in that fixed to the bottom of the reagent chamber (surface of the sealing film).

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