KR20140085710A - Micro chip for detecting food-borne bacteria, real-time PCR device comprising the same, and method for detecting food-borne bacteria using the same - Google Patents

Abstract

An embodiment of the present invention relates to a micro PCR chip and a real-time PCR device comprising the same. According to the embodiment, provided are a PCR chip which can rapidly obtain results by accommodating a plurality of small samples at the same time and maximally obtaining thermal contact efficiency with thermal blocks and can accurately measure light signals emitted from nucleic acid amplification products without separate filtering or processing; and a real-time PCR device which can rapidly obtain reliable nucleic acid amplification results without a complex light signal measuring module.

Description

TECHNICAL FIELD The present invention relates to a micro-PCR chip including a primer set for detecting food poisoning, a real-time PCR apparatus including the same, and a method for detecting food poisoning using the same, borne bacteria using the same}

The present invention relates to a micro PCR chip including a primer set for the detection of food poisoning bacteria, a real time PCR apparatus including the same, and a food poisoning detection method using the same.

Foodborne pathogens are spread mainly through food such as meat, dairy products, and drinking water. Therefore, methods for rapidly and economically confirming the presence of foodborne pathogens in food-like samples are required. A common method for detecting food poisoning bacteria is to culture a sample in a selective medium to isolate bacteria suspected to be food poisoning bacteria, and then to identify them by biochemical or immunological methods. However, an immunological method using an antibody can detect bacteria with high accuracy, but a large amount of sample is required. In order to produce an antibody necessary for each diagnosis, protein purification, production or peptide production of the bacterium is essential, High antibody production costs are required. In addition, due to the nature of the protein, there are many difficulties in storage and utilization, and only one kind or a limited number of kinds of bacteria can be detected at a time, and a long time is consumed in the cultivation and experimental steps of the bacteria. To overcome these disadvantages, various bacterial detection kits using PCR method have been researched and developed. Detection kits using the PCR method have been increasingly demanded in various fields because of their high accuracy, simplicity and promptness.

In particular, the real-time PCR method, which is widely used in recent years, is a method to observe the increase of the PCR amplification product in real time in every cycle of the PCR and to detect and quantify the fluorescent substance reacting with the PCR amplification product. This method eliminates the need for additional electrophoresis, has excellent accuracy and sensitivity, has high recall, and can be automated, as compared with conventional PCR method after completion of the final step and staining on gel to confirm PCR amplification products after electrophoresis And the result can be quantified, and it is quick and easy, and it is excellent in biological safety due to harmful problems such as contamination by EtBr (Ethidium Bromide) and irradiation with ultraviolet ray, and it is possible to automatically check whether the specific gene is amplified There are advantages. Thus, quantitative results with high specificity, not qualitative results such as PCR or antigen / antibody, can be identified through real-time PCR methods. In addition, since the probe labeled with a fluorescent marker is used, the result can be confirmed even with a sample smaller than the amount of the sample used for the DNA chip or the antigen / antibody reaction. Therefore, there is a need to develop a detection method and detection kit for food poisoning bacteria using a real-time PCR method in order to quickly and accurately diagnose the infection of food poisoning bacteria in foods.

Real-time polymerase chain reaction (PCR) is widely used in recent nucleic acid analysis because it can be confirmed in real time during a reaction cycle without electrophoresis on a gel. In general, an apparatus for real-time PCR includes a thermal cycler having one or more heating blocks for performing a nucleic acid amplification reaction and a signal for measuring a signal generated from the nucleic acid amplification product in real time Detector. Such a signal detector is exemplified by a photodetector for detecting a fluorescence signal generated from a nucleic acid amplification product, an electrical signal detector for detecting an electrical signal generated through a specific combination of a nucleic acid amplification product and a mutual coupling medium .

Recently, in the field of medical care, efficient diagnosis and treatment methods for implementing customized medicine are being actively developed. In order to realize customized medicine, rapid and accurate diagnosis and treatment of a large number of individuals are required. In this case, in the diagnosis and treatment, the nucleic acid amplification step is the most basic preconditioning step, and real-time PCR, which is an example of performing this, is a preconditioning step in the realization of customized medicine. However, since real-time PCR requires a complicated process, it takes a considerable time to complete the PCR, and the device for realizing the PCR is expensive and large, which hinders realization of customized medicine. Many attempts have been made recently to solve such problems.

In this regard, Korean Patent Laid-Open No. 10-2004-0048754 (temperature-controlled real-time fluorescence detection apparatus) is capable of detecting fluorescence of several wavelengths in several hundreds to thousands of samples in a few seconds at a fast and low sample concentration And provides a compact fluorescent search device that can search and analyze enzyme reactions in real time and can be carried at an economical price. Specifically, the preceding fluorescence detection apparatus is an apparatus for analyzing a sample by irradiating a biological sample with a light source and then detecting fluorescence emitted from the sample, the apparatus comprising: a sample container; a light source positioned to irradiate the sample container; CLAIMS What is claimed is: 1. A fluorescence detection apparatus comprising: a detector for detecting fluorescence; a fluorescence transfer apparatus for transferring fluorescence emitted from the sample to the detector; a wavelength selector; and a control unit, the apparatus comprising: an LED array arranged to sequentially emit a plurality of LEDs; A well chamber block having a plurality of wells for inserting a sample container; A multi-channel PMT for detecting fluorescence emitted from the sample by each LED emission of the LED array; And a plurality of optical fibers for moving the fluorescence emitted from each sample individually to the multi-channel PMT.

In addition, Korean Patent No. 10-0794703 (a real-time monitoring apparatus for biochemical reactions) provides an apparatus capable of comparing and analyzing the reaction degree of various kinds of samples by minimizing the optical detection sensitivity variation in the reaction in the reaction tube plate. Specifically, the preceding real-time monitoring apparatus includes a temperature control block system including a thermoelectric element for supplying heat to the reaction tube and a heat transfer block for transferring heat to the reaction tube; An irradiating light source unit including a lamp and an optical waveguide for irradiating uniformly light to the sample in the reaction tube; And an optical system including a reflecting mirror for changing the optical path and a light receiving unit for receiving fluorescence generated in the sample in the reaction tube by the light irradiated by the irradiation light source unit.

Korean Patent No. 10-1089045 (a real-time monitoring device for nucleic acid amplification reaction product) is capable of performing a nucleic acid amplification reaction such as a polymerase chain reaction and monitoring the generation of reaction products generated in the reaction in real time A real-time monitoring apparatus for a biochemical reaction including a polarizer, a polarization beam splitter, a polarization converter, and the like is provided for efficiently separating interference between excitation light and fluorescence.

In addition, Korean Patent Laid-Open No. 10-2008-0103548 (a real-time detection apparatus for nucleic acid amplification products) is designed so as to effectively eliminate or reduce the error factors on the apparatus without using the second fluorescence signal used for correction, Temperature cycles are applied to a plurality of wells to detect the fluorescence intensity from the nucleic acid amplification product in each well in real time, and furthermore, the fluorescence measured value [DNA] raw obtained from the well and the peripheral connection The fluorescence intensity [DNA] real of the well can be determined by detecting the fluorescence measurement value [DNA] bg obtained from the wall and subtracting the fluorescence measurement value [DNA] bg from the fluorescence measurement value [DNA] Detection device.

Korean Patent No. 10-0794699 (a real-time monitoring apparatus for nucleic acid amplification reaction products) is capable of performing nucleic acid amplification reactions such as polymerase chain reaction in a very small amount of samples and monitoring the generation of reaction products generated in the reaction in real time A sample reaction unit comprising a reaction vessel having a plurality of wells for containing a plurality of samples, a transparent sealing cover for covering the reaction vessel, and a thermoelectric element for supplying a heat source to the reaction vessel; A selective transmission filter located in front of the excitation light source, and a linear polarization element for linearly polarizing the light passing through the filter; A line polarizer in a direction perpendicular to the line polarizer of the light emitting element, a condenser lens for condensing the light passing through the linear polarizer, a selective transmission filter for selectively transmitting the light passing through the condenser lens, and a light receiving element unit A nucleic acid amplification reaction product, and a nucleic acid amplification reaction product.

However, since the above-mentioned prior arts add a complicated and elaborate fluorescent signal measurement module to simultaneously measure a plurality of nucleic acid amplification products, problems of large-sized and high-cost devices are still a problem. Furthermore, although the above prior art attempts to simultaneously measure a small number of samples, the signal sensitivity is significantly reduced by a bubble generated by heating in a small amount of sample solution contained in a small reaction container during the nucleic acid amplification process There is no solution for the phenomenon of the above-mentioned problems.

Therefore, there is still a need for a real-time PCR implementation device capable of simultaneously measuring a small number of nucleic acid amplification products and ensuring the reliability of the measured values, and further realizing rapid monitoring of nucleic acid amplification products at low cost, The present invention can be applied to a detection device capable of simultaneously and rapidly detecting food-borne bacteria in food, and a method for detecting food-borne bacteria using the same.

The present invention utilizes a micro PCR chip capable of simultaneously measuring a small number of small amounts of nucleic acid amplification products, detecting nucleic acid amplification products at low cost, and further ensuring the reliability of the results. The present invention provides a food poisoning detection device and a detection method which can simultaneously and quickly detect a food poisoning.

In order to accomplish the above-mentioned problem to be solved,

One embodiment of the present invention includes at least 16 PCR reaction chambers having open top faces; And a plurality of protrusions protruding toward the inside of the PCR reaction chamber from a part of the sealing surface contacting the open top face to abut the open top face of the PCR reaction chamber to seal the open top face, A cover having a light transmitting portion of a material; Wherein the primer comprises at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2 in the 16 or more PCR reaction chambers. Listeria monocytogenes iap a primer set for detecting an invasion associated protein gene; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer for detecting the nucA (nuclease) gene of Staphylococcus aureus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: set; A primer comprising SEQ ID NO: 6 and primer nucleotide sequence 15 or more consecutive nucleotides of a nucleotide sequence containing at least 15 consecutive nucleotides of the SEQ ID NO: 5 consisting of Shigella spp . A primer set for detecting an ipaH (invasive plasmid antigen gene) gene; A primer set for detecting a toxR gene of Vibrio parahaemolyticus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 8; A primer comprising SEQ ID NO: 9 of the base sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 10 nucleotide sequence at least 15 consecutive nucleotides of a Bacillus comprising enterotoxin of cereus FM a primer set for detecting an entFM gene; Salmonella consisting of a primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 12 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 11 spp . A primer set for detecting an invA protein of an invA ; A primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 14 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 13 consisting of Yersinia a primer set for detecting the ail (attachment invasion locus) gene of enterocolitica ; Clostridium consisting primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the Primer set for detecting the cpe (C. perfringens enterotoxin) gene of perfringens; A primer comprising SEQ ID NO: 17 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 18 nucleotide sequence at least 15 consecutive nucleotides of the made Campylobacter a primer set for detecting the hypo gene of jejuni ; ( LT ) of Enterotoxigenic E. coli ( ETEC ) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 19 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 20 a primer set for detecting an enterotoxin gene; STX1 of Enterohemorrhagic E. coli (EHEC) consisting of a primer comprising SEQ ID NO: 22 and a primer of the nucleotide sequence at least 15 consecutive nucleotides of a nucleotide sequence comprising at least 15 consecutive nucleotides of the SEQ ID NO: 21 (shiga-like a primer set for detecting a toxin-1 gene; Detecting the eaeA gene of Enteropathogenic E. coli ( EPEC ) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set; Detecting the invA gene of Enteroinvasive E. coli ( EIEC ) comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 25 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 26 Primer set; A primer containing the primers and SEQ ID NO: 28 nucleotide sequence at least 15 consecutive nucleotides of the nucleotide sequence containing at least 15 consecutive nucleotides of the SEQ ID NO: 27 consisting Camphylobacter a primer set for detecting the ceuE gene of E. coli ; Vibrio made of a primer comprising SEQ ID NO: 29 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 30 nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the ompW gene of cholera ; And Vibrio consisting of a primer comprising the base sequence of SEQ ID NO: 31 primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 32 and a nucleotide sequence at least 15 consecutive nucleotides of the and a primer set for detecting a Vvh gene of a vulnificus , and a primer set for detecting a Vvh gene of a vulnificus .

According to an embodiment of the present invention,

The PCR reaction chamber may be implemented to have a liquid sample capacity of 10 μl or less. In this case, the PCR reaction chamber can accommodate 5 to 8 [mu] l of liquid sample.

The light transmitting portion may be disposed at the center of the closed surface.

The light transmitting portion may be formed to reach the bottom surface of the PCR reaction chamber or to a position spaced upwards from the bottom surface of the PCR reaction chamber.

The cover may further include a hole passing through the light transmitting portion and a flexible packing portion contacting the open top face of the PCR reaction chamber to seal the open top face.

The micro PCR chip for detecting food poisoning bacteria may be implemented to have a flat plate shape.

The micro PCR chip for detecting food poisoning bacteria comprises a first plate having a flat plate shape; A second plate disposed on the first plate and having a PCR reaction chamber; And a third plate disposed on the second plate to seal the open top face of the PCR reaction chamber and serve as a cover having the light transmitting portion, . In this case, a hole is formed between the second plate and the third plate so as to penetrate the light transmitting portion, and a flexible packing portion that abuts the open top surface of the PCR reaction chamber to seal the open top surface can do.

And a heat discharging unit configured to discharge heat generated from the PCR reaction chamber to the outside.

Another embodiment of the present invention is a micro PCR chip for detecting the food poisoning bacteria; At least one thermal block arranged to be in thermal contact with at least one surface of the micro-PCR chip for detecting food poisoning bacteria; And an optical detection module implemented to detect an optical signal generated from the PCR amplification product inside the PCR reaction chamber of the micro-PCR chip for detecting food poisoning bacteria.

Another embodiment of the present invention is a micro PCR chip for detecting the food poisoning bacteria; A first column block disposed on the substrate and configured to be in thermal contact with the micro PCR chip; A second thermal block disposed on the substrate and spaced apart from the first thermal block and configured to be in thermal contact with the micro PCR chip; A chip holder mounted on the first column block and the second column block, the chip holder being movable in a lateral direction and / or vertically by a driving means, the micro PCR chip mounted; And a micro PCR chip for detecting the food poisoning bacteria is disposed between the first column block and the second column block, and when the micro PCR chip for detecting food poisoning bacteria is moved between the first column block and the second column block by the driving unit, And a photodetector module configured to detect an optical signal generated from the PCR amplification product of the PCR amplification product.

According to the above-mentioned problem solving means, a micro PCR chip capable of rapidly measuring a small number of nucleic acid amplification products simultaneously, detecting nucleic acid amplification products at a low cost, and further securing the reliability of results can be provided The present invention can provide a food poisoning detection device and a detection method capable of rapidly and accurately detecting 16 kinds of food poisoning bacteria at the same time.

FIGS. 1 to 3 relate to a phenomenon in which optical signal sensitivity is reduced by a bubble generated during the PCR process in a PCR container (small size, x 1/20) in a polarized-miniaturized PCR container (large size).
4 is a cross-sectional view of a basic configuration of a micro PCR chip according to an embodiment of the present invention.
FIG. 5 is a diagram illustrating a principle in which an optical signal is released from a PCR product without being affected by a bubble generated during a PCR process in a micro PCR chip according to an embodiment of the present invention.
6 illustrates various types of light transmitting portions of a micro PCR chip according to an embodiment of the present invention.
7 to 9 relate to a flexible packing part of a micro PCR chip according to an embodiment of the present invention.
10 is a micro PCR chip according to an embodiment of the present invention in which two or more unit modules including a PCR reaction chamber and a cover having a light transmission portion are repeatedly implemented.
11-12 are cross-sectional exploded views of a micro PCR chip according to an embodiment of the present invention.
13 is a micro PCR chip according to an embodiment of the present invention including a heat releasing part.
FIGS. 14 to 15 are schematic diagrams for explaining a method for detecting an optical signal generated from a PCR amplification product inside a PCR reaction chamber of a micro PCR chip, a thermal block in thermal contact with the micro PCR chip, and a micro PCR chip according to an embodiment of the present invention And a real-time PCR apparatus according to another embodiment of the present invention including the implemented photodetection module.
16 to 18 are schematic diagrams showing a micro-PCR chip, two column blocks, a chip holder mounted with the micro-PCR chip, movable between the two column blocks by a driving means, Wherein the micro PCR chip is configured to detect an optical signal generated from a PCR amplification product inside a PCR reaction chamber of the micro PCR chip when the driving unit moves between the two column blocks, , And a real-time PCR apparatus according to another embodiment of the present invention.
19 is an actual implementation diagram of a micro PCR chip according to an embodiment of the present invention.
20 is an electrophoresis image obtained by performing PCR using a micro PCR chip according to an embodiment of the present invention including a primer set for 16 kinds of food poisoning bacteria.
FIG. 21 is a table showing 16 food poisoning bacteria corresponding to the electrophoresis photographs of FIG. 20.

Hereinafter, embodiments according to the present invention will be described in detail with reference to the accompanying drawings. The following description is merely intended to facilitate understanding of embodiments of the present invention and is not intended to limit the scope of protection of the present invention.

An embodiment of the present invention relates to a polymerase chain reaction (PCR), and more particularly, real-time PCR for monitoring nucleic acid amplification reaction in real time.

PCR is a technique for repeatedly heating and cooling a PCR sample and a reagent containing a nucleic acid to sequentially amplify a specific base sequence region of the nucleic acid and amplify the nucleic acid having the specific base sequence region exponentially. It is widely used for diagnosing and analyzing diseases in engineering and medical fields. PCR apparatuses for efficiently performing PCR have recently been developed variously. A PCR apparatus collectively refers to a device implemented to perform PCR to amplify a nucleic acid having a specific nucleotide sequence. Generally, a PCR apparatus is a denaturing step in which PCR samples and reagents containing double stranded DNA are heated to a specific temperature, for example, about 95 DEG C to separate the double stranded DNA into single stranded DNA, An oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified is provided in the PCR sample and the reagent, and the oligonucleotide primer is cooled with a single strand DNA to a specific temperature, for example, An annealing step of binding the primer to a specific nucleotide sequence of the strand DNA to form a partial DNA-primer complex, and, after the annealing step, the PCR sample and the reagent are incubated at an activity temperature of the DNA polymerase, And maintained at 72 ° C to form a double stranded DNA based on the primer of the partial DNA-primer complex by a DNA polymerase DNA amplification can be performed exponentially by performing an extension (or amplification) step and repeating the extension (or amplification) step, for example, 20 to 40 cycles . In the meantime, a recent 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 the annealing and extension (or amplification) steps following the denaturation step , Thereby completing the first circulation.

In real-time PCR, an optical system module such as a measuring device, for example, a fluorescence photometer is applied to a thermal cycler used for PCR to perform a nucleic acid amplification reaction capable of monitoring the generation process of the nucleic acid amplification product It says. Unlike conventional PCR, real-time PCR does not require electrophoresis for confirmation of nucleic acid amplification products, so that nucleic acid amplification products can be analyzed in real time in an accurate and rapid manner. Recently, a real-time PCR apparatus has been actively developed. In order for the real-time PCR apparatus to fully exhibit the above advantages, it is necessary not only to increase the efficiency of the thermocycler, but also to accurately measure the optical signal generated from the nucleic acid amplification product Should be able to do.

FIGS. 1 to 3 relate to a phenomenon in which optical signal sensitivity is reduced by a bubble generated during a PCR process in a conventional PCR container (large size) and an extremely small-sized PCR container (small size, x 1/20).

For practical realization of customized medical services, the recent PCR apparatuses are aimed at miniaturization, portability, promptness, and economy. Conventional PCR devices were not only difficult to carry, difficult to carry, as well as containers for PCR samples and reagents, but also the PCR samples and reagents were wasted, resulting in considerable cost. Furthermore, since the amount of the PCR sample and the reagent to be used is large, it takes a considerable time and it is difficult to realize efficient PCR.

1, the left side is a conventionally used PCR container (large size), and the right side is a PCR container (size: small) (x1 / 20) in which the size of the PCR container ). Conventionally, a conventional PCR container (large-size) is composed of a reaction chamber containing a PCR sample and a reagent and a cover thereof, the reaction chamber and the cover are made of a light-transmitting material, and about 200 [ PCR was carried out with a sample volume of about 20 μl of sample and reagent. The PCR container (small size) is also composed of a reaction chamber containing a PCR sample and a reagent, and a cover of the reaction chamber. The reaction chamber and the cover may be formed of a light-transmitting material. In this case, Small) has a liquid sample capacity of about 10 [mu] l, and the PCR is carried out with about 5 to 8 [mu] l of the sample and the reagent being accommodated. Thus, the fabrication of ultra-miniaturized PCR vessels can be readily implemented in the currently known art. However, miniaturization of the PCR container is difficult to implement because it has a considerable adverse effect in the measurement of the nucleic acid amplification product as described below.

Referring to FIG. 2, it can be easily confirmed that the optical signal sensitivity is reduced due to a bubble generated during the PCR process in a PCR container (small size, x 1/20) which is smaller than the conventional PCR container . As described above, since the PCR involves a heat supply step, a considerable amount of bubbles is generated inside the PCR container due to the heating of the liquid sample, which blocks the light signal generated from the nucleic acid amplification product do. 2, the bubbles generated in the PCR container (large-sized) reduce the optical signal sensitivity by blocking the optical signal generated from the nucleic acid amplification product, The bubbles are dispersed inside the PCR container (large size) or form a cluster on the inner wall of the PCR container (large size), so that the optical signal measurement is not impossible even though the optical signal sensitivity is decreased. However, according to FIG. 3, which enlarges the "a" portion of FIGS. 2 and 2, the bubbles generated inside the PCR container (compact) have a considerably small internal space of the reaction container, The optical signal generated from the nucleic acid amplification product is blocked, resulting in a considerable decrease in the optical signal sensitivity and nonuniformity, resulting in a lower reliability of the result. Therefore, in order to miniaturize the PCR device and to realize the miniaturization of the PCR container mounted on the PCR device, it is necessary to sufficiently consider the reduction of the optical signal sensitivity and securing the reliability of the result according to the unevenness.

4 is a cross-sectional view of a basic configuration of a micro PCR chip according to an embodiment of the present invention.

Referring to FIG. 4, a micro-polymerase chain reaction chip 1 according to an embodiment of the present invention includes a PCR reaction chamber 10 having a top open; And an upper end surface of the PCR reaction chamber (10) to seal the upper open end surface, and protrudes from a part of the closed surface contacting the open top surface toward the inside of the PCR reaction chamber (10) And a cover 20 having a light transmitting portion 25 of a light transmitting material extending along the light emitting portion 21.

The PCR reaction chamber 10 is opened to receive the liquid sample, that is, the PCR sample and the reagent, while the upper surface is opened and the lower surface and the side surface are sealed. The PCR reaction chamber 10 should be implemented so as not to be affected by repetitive heating and cooling during the PCR process, and is not limited to a specific shape and / or material as long as such function can be maintained. However, since the micro PCR chip 1 according to an embodiment of the present invention is based on real-time optical signal measurement of the nucleic acid amplification product, it is preferable that at least a portion overlapping the optical path 21 is realized by a light transmitting material Do.

The cover 20 abuts against the open top surface of the PCR reaction chamber 10 to seal the open top surface. As the lid 20 closes the open upper end surface of the PCR reaction chamber 10, the PCR sample and reagent reacting inside the PCR reaction chamber 10 are not leaked to the outside, and the PCR reaction chamber 10, It serves to maintain the internal temperature. Meanwhile, the lid 20 may be embodied in various shapes and / or materials as long as the above functions can be realized. However, since the micro PCR chip 1 according to an embodiment of the present invention is based on real-time optical signal measurement of the nucleic acid amplification product, it is preferable that the micro PCR chip 1 is implemented as a light transmitting material.

4, the lid 20 protrudes toward the inside of the PCR reaction chamber 10 from a part of the closed surface contacting the open top face, and is formed of a light transmitting material extending along the optical path 21 The light transmitting portion 25 of the light guide plate 25 is provided. The light transmitting portion 25 is implemented as a light transmitting material and is extended along the optical path 21 for measurement of the nucleic acid amplification product. The optical signal generated from the nucleic acid amplification product inside the PCR reaction chamber 10 passes through . The light transmitting portion 25 is provided on the sealing surface contacting the open top face of the PCR reaction chamber 10, that is, from a part of the bottom face of the cover 20 toward the inside of the PCR reaction chamber 10 Downwardly protruding. The projecting shape of the light transmitting portion 25 may vary, but is preferably formed in a cylindrical or square pillar shape. 6, the protruding shape of the light transmitting portion 25 can be variously realized, and it can be realized to touch the bottom surface of the PCR reaction chamber 10 (right side of FIG. 6) To a position spaced upwardly from the bottom bottom surface of the chamber 10 (left side in FIG. 6). That is, the light transmitting portion 25 may be adjacent to or in contact with the surface of the liquid sample when the liquid sample is received in the PCR reaction chamber 10, or may be contained in the liquid sample through the liquid sample surface have. In addition, if the light transmitting portion 25 is implemented to extend along the optical path, the light transmitting portion 25 may be provided on the sealing surface contacting the open top surface of the PCR reaction chamber 10, It is preferable that the cover 20 is disposed at the center of the closed surface, that is, at the central area of the lower end surface of the cover 20. On the other hand, it is preferred that the liquid sample capacity of the PCR reaction chamber 10 is not limited to a specific volume but is implemented to have a liquid sample capacity of 10 μl or less to accommodate 5 to 8 μl of liquid sample.

FIG. 5 is a diagram illustrating a principle in which an optical signal is released from a PCR product without being affected by a bubble generated during a PCR process in a micro PCR chip according to an embodiment of the present invention.

As the PCR process proceeds, the liquid sample in the PCR container may be heated and bubbles may be generated as described above.

5, when a liquid sample in the PCR reaction chamber 10 of the micro PCR chip 1 according to an embodiment of the present invention, that is, the PCR sample and the reagent, is heated by the heat supply Bubbles occur. However, in the case of the micro PCR chip 1 according to an embodiment of the present invention, a portion of the closed surface protruding from the lower end surface of the cover 20, that is, the open top surface of the PCR reaction chamber 10 The PCR reaction chamber 10 is irradiated with light from the light transmitting portion 25 protruding from the center region of the PCR reaction chamber 10 toward the inside of the PCR reaction chamber 10 and extending along the optical path 21 The formed bubble is pushed to the peripheral region of the rim surface of the light transmitting portion 25 and is compressed and arranged in the peripheral space. Accordingly, the bubble is completely deviated from the optical signal path (light transmitting portion) 25 formed from the nucleic acid amplification product present in the liquid sample, and the optical signal sensitivity for measuring the nucleic acid amplification product It has no effect at all. Therefore, when the nucleic acid amplification product is measured in real time during the real-time PCR process using the micro PCR chip 1 according to an embodiment of the present invention, the influence of the bubble generated inside the PCR reaction chamber 10 The optical signal sensitivity is significantly increased. As a result, according to the micro-PCR chip 1 according to the embodiment of the present invention, since the capacity of the liquid sample can be greatly reduced to, for example, 10 μl or less compared with the conventional PCR container, the PCR container can be miniaturized And the optical signal sensitivity can be significantly increased at the same time, it is possible to achieve miniaturization and portability of the PCR container and the real-time PCR device, and furthermore, it is possible to rapidly and accurately measure a plurality of small amounts of nucleic acid amplification products simultaneously .

7 to 9 relate to a flexible packing part of a micro PCR chip according to an embodiment of the present invention.

7 to 9, the lid 20 of the micro PCR chip 1 according to an embodiment of the present invention includes a hole 45 passing through the light transmitting portion 25, And a flexible packing portion 40 which abuts against the open top face of the chamber 10 to seal the open top face.

The flexible packing unit 40 prevents leakage of a liquid sample due to a bubble or a pressure rise due to a temperature rise in the PCR reaction chamber 10 during the PCR process. The flexible packing part 40 is made of a material such as rubber or silicon having elasticity or elasticity to buffer the expansion force due to the generation of bubbles or pressure, So that it can be maintained in an airtight state. Although the holes 45 are implemented according to the shape of the light transmitting portion 25, they are not limited thereto. 8 shows a state in which the flexible packing part 40 is attached to the lid 20 and surrounds the light transmitting part 25 and Fig. 9 shows a state in which the lid 20 in the state of Fig. 8 And is coupled to the upper surface of the PCR reaction chamber 10 to seal the inner space of the PCR reaction chamber 10.

10 is a micro PCR chip according to an embodiment of the present invention in which two or more unit modules including a PCR reaction chamber and a cover having a light transmission portion are repeatedly implemented.

As described above, the micro PCR chip 1 according to an embodiment of the present invention significantly increases the optical signal sensitivity by the lid 20 having the PCR reaction chamber 10 and the light transmitting portion 25 It is possible to realize a PCR container having a multi-chamber structure in which a plurality of small liquid samples can be accommodated without being influenced or minimized.

Referring to FIG. 10, the micro PCR chip 1 according to an embodiment of the present invention may include two or more unit modules 50 including the PCR reaction chamber 10 and the cover 20. For example, as shown in FIG. 10, when the micro PCR chip 1 is implemented in a flat plate shape, the unit modules 50 may be arranged in a line or integrated into a circular space on a flat plate, For example, the unit module 50 may be implemented as 19 wells, 48 wells, or 96 wells.

11-12 are cross-sectional exploded views of a micro PCR chip according to an embodiment of the present invention.

Referring to FIG. 11, a micro PCR chip 1 according to an embodiment of the present invention includes a first plate 100 of a flat plate shape; A second plate (200) disposed on the first plate (100) and having a PCR reaction chamber (10); And a cover (20) disposed above the second plate (200), wherein the cover (20) is provided with the light transmitting portion (25) by abutting the open top face of the PCR reaction chamber (10) And a third version 300 that plays a role.

The first plate 100 is implemented in a flat plate shape and serves as a bottom support of the micro PCR chip 1 according to an embodiment of the present invention. The first plate 100 may be formed of various materials. However, the first plate 100 may be formed of a plastic material such as polycarbonate (PC), polyethylene terephthalate (PET) And is preferably made of a transmissive material. In addition, although the surface of the first plate 100 may be variously embodied, it is preferable that the surface of the first plate 100 is treated to have a hydrophilic surface. In addition, the first plate 100 may preferably be implemented at about 0.03 to 1.0 mm, and more preferably at about 0.1 to 0.5 mm.

The second plate 200 is formed in a flat plate shape and is disposed on the first plate 100. The second plate 200 is disposed on the upper side of the PCR reaction chamber 10 of the micro PCR chip 1 according to an embodiment of the present invention. . The second plate 200 may be formed of various materials. However, the second plate 200 may be formed of a plastic material such as polycarbonate (PC), polyethylene terephthalate (PET) And is preferably made of a transmissive material. In addition, the second plate 200 may preferably be implemented at about 0.5 to 5 mm, and more preferably at about 1 to 2 mm.

11, a bottom surface space of the PCR reaction chamber 10 of the micro PCR chip 1 according to an embodiment of the present invention is disposed between the first plate 100 and the second plate 200 An additional layer 150 in the form of a flat plate to be formed may be formed. This may be a bonding surface between the first plate 100 and the second plate 200, or it may be an adhesive layer. Accordingly, the first plate 100 and the second plate 200 may be bonded by thermal bonding, ultrasonic bonding, ultraviolet bonding, or solvent bonding. In addition, the additional layer 150 may preferably be implemented at about 0.03 to 1.0 mm, and more preferably at about 0.1 to 0.5 mm.

The third plate 300 is formed in a flat plate shape and is disposed on the upper side of the second plate 200. The third plate 300 can be opened and closed in the PCR reaction chamber 10 of the micro PCR chip 1 according to an embodiment of the present invention. And serves as a lid 20 having the light transmitting portion 50 to close the open top face by abutting against the top face. The third plate 200 may be formed of various materials. However, the third plate 200 may be formed of a plastic material such as polycarbonate (PC), polyethylene terephthalate (PET) And is preferably made of a transmissive material. In addition, the third plate 200 may preferably be implemented at about 0.5 to 5 mm, and more preferably at about 1 to 2 mm.

12, the third plate 300 may include a hole between the second plate 200 and the third plate 300 to surround the light transmitting portion 25, And a flexible packing part 40 which abuts on the open top face of the reaction chamber 10 to seal the open top face. The flexible packing unit 40 serves to prevent leakage of the PCR sample and the reagent contained in the PCR reaction chamber 10 and contamination between the plurality of chambers. The flexible packing unit 40 may be made of various materials having flexibility or stretchability, but is preferably formed of, for example, silicon, telflon, or the like. In addition, the flexible packing portion 40 may preferably be embodied as about 0.1 to 2 mm, more preferably about 0.5 to 1 mm, and the circular hole diameter is preferably about 1.0 mm . ≪ / RTI >

13 is a micro PCR chip according to an embodiment of the present invention including a heat releasing part.

The micro PCR chip 1 according to an embodiment of the present invention may further include a heat emitting unit 60 configured to emit heat generated from the PCR reaction chamber 10 to the outside. Referring to FIG. 13, the micro PCR chip 1 according to an embodiment of the present invention is formed as a whole with a thin plate shape, and a plurality of unit modules 50 are integrated in a central circular area. As described above, since high temperature heat is generated in the PCR reaction chamber 10 in the unit module 50 during the PCR process, the micro temperature of the microchannel according to one embodiment of the present invention, The PCR chip 1 can dispose the heat releasing part 60 on both sides of the central circular area.

14 to 15 show a real-time PCR apparatus having a single column block to which a micro PCR chip according to an embodiment of the present invention is applied.

14 to 15, a real-time PCR apparatus 2000 according to another embodiment of the present invention includes a micro-PCR chip 1 according to an embodiment of the present invention as described above; At least one thermal block (200) implemented to be in thermal contact with at least one side of the micro PCR chip (1); And an optical detection module 300 configured to detect an optical signal generated from the PCR amplification product inside the PCR reaction chamber 10 of the micro PCR chip 1.

The thermal block 200 is a module that is thermally contacted with the micro PCR chip 1 to enable heat exchange. The thermal block 200 may be implemented in a variety of materials and may be implemented to be totally (or partially) transmissive to measure an optical signal of a nucleic acid amplification product. The transparent heat-generating material may include any material that has transparency and is exothermic due to power supply, but preferably includes indium tin oxide (ITO), a conducting polymer, a carbon nano- And may be selected from the group consisting of a tube (Cabon NanoTube, CNT), graphene, a transparent conductive oxide (TCO), and an oxide-metal-oxide multilayer transparent element. Indium tin oxide (ITO) is a mixture of indium oxide (In ₂ O ₃ ) and tin oxide (SnO ₂ ), and is generally composed of 90% of indium oxide and 10% of tin oxide, Or ITO. Indium tin oxide, when implemented as a thin layer, has electrical conductivity, is transparent, has no color, and is yellowish when it is embodied in a lump state. Indium tin oxide is deposited on the surface of other materials by electron beam evaporation, vapor deposition, and sputtering techniques. Indium tin oxides are typically used in liquid crystal displays, flat panel displays, plasma displays, touch screens, electronic paper, , Antistatic coatings, and electron blocking coatings. A conducting polymer is called a so-called electrically conductive plastic, and has an advantage of being excellent in light transmittance, light in weight, excellent in elasticity and electric conductivity, and easy to be processed. Conductive polymers are made of materials such as polyacetylenes, polypararylene, polyphenols, polyanilines and the like, and recently, they are sometimes produced from polystyrene sulfonic acid and / or PEDOT (poly (3,4-ethylenedioxythiophene)). Cabon NanoTube (CNT) refers to a fine molecule with a diameter of 1 nanometer, which is shaped like a long barrel of carbon atoms connected by hexagonal rings. It is known that tensile strength is stronger than steel, has excellent flexibility, is lightweight, and has high electrical conductivity. On the other hand, when a purified single-walled carbon nanotube (SWNT) is dispersed in a solvent using a surfactant and formed by using a vacuum filter device, a transparent conductor is formed, which has both transparency and conductivity do. Graphene is a nanomaterial composed of carbon, such as carbon nanotubes and fullerene, which is an atomic number 6 carbon, separated from graphite in the early 2000s. Graphene is known to have a high electrical conductivity and superior elasticity, more than 100 times more than copper, and has recently been used as a transparent electrode for various applications. Transparent conductive oxide (TCO) refers to a substance having transparency among various metal oxides combined with oxygen, and includes ZnO, SnO ₂ , TiO ₂ , and the like. Transparent metal oxides have high conductivity and transparency and can be used as coating materials at low cost. The oxide-metal-oxide multilayer transparent device is manufactured by a roll-to-roll sputtering process, and can be realized with flexibility, low resistance, and high transmittance of oxide of ITO-Ag (or Cu) -ITO, AZO- AZO, GZO-Ag-GZO, IZO-Ag-IZO and IZTO-Ag-IZTO. 14 to 15, the thermal block 200 may be implemented in various shapes, but is preferably implemented in a flat plate shape. The thermal block 200 in the form of a flat plate can provide uniform heat to the mixed solution of the PCR sample and the reagent because the surface area of the micro-PCR chip 1, preferably in contact with the plate-shaped chip, is wide, The temperature change of each cycle can proceed rapidly. On the other hand, in order to accurately monitor real-time PCR products, it is necessary to increase the sensitivity of the optical signal as much as possible. The thermal block 200 may be implemented to have optical transparency as a whole, so that most of the excitation light emitted from the light source can be transmitted as it is to increase the optical signal sensitivity. However, a part of the excitation light may be reflected on the column block 200 or may be reflected after passing through the column block 200 to act as noise of the optical signal. Accordingly, preferably, the lower surface of the thermal block 200 may be treated with a light absorbing material to further increase the optical signal sensitivity. The light absorbing substance may be, for example, mica, but is not limited as long as it is a substance having a property of absorbing light. Therefore, the light absorbing layer absorbs a part of the light derived from the light source, and the generation of the reflected light acting as noise of the optical signal can be suppressed as much as possible. Alternatively, the top surface of the thermal block 200 may be treated with a light reflection preventing material to further increase the optical signal sensitivity. The light reflection preventing material may be, for example, a fluoride such as MgF ₂ , an oxide such as SiO ₂ or Al ₂ O ₃ , but is not limited as long as it is a material capable of preventing light reflection. Further, more preferably, the optical signal sensitivity can be further improved by processing the light absorbing material on the lower surface of the thermal block 200 and treating the upper surface of the thermal block 200 with a light reflection preventing material. That is, in order to effectively monitor the real-time PCR, the ratio of the optical signal to the noise should have a maximum possible value, and the ratio of the optical signal to the noise can be improved as the reflectance of the excitation light from the PCR chip is low. For example, when the heat block 200 including the light absorbing layer or the light reflection preventing layer is used, the reflectance of the excitation light of the conventional heat block of a general metallic material is about 20 to 80% %, And when the heat block 200 including the light absorbing layer 60 and the light reflection preventing layer 70 is used, the light reflectance can be reduced to 0.2% or less.

The photodetection module 300 is provided with a photodetector (not shown) operatively arranged to provide light to the micro PCR chip 1 and a photodetector (not shown) operable to receive light emitted from the micro PCR chip 1 (Not shown) disposed on the substrate. The light providing unit is a module for providing light to the micro PCR chip 1. The light receiving unit receives the light emitted from the micro PCR chip 1 and generates a PCR product . Light is emitted from the light provider and the emitted light passes through or reflects the PCR reaction chamber in the micro PCR chip 1, specifically, the unit module 50 of the micro PCR chip 1. In this case, And the optical detection unit detects an optical signal generated by nucleic acid amplification in the PCR reaction chamber. Therefore, according to the real-time PCR apparatus 1000 according to another embodiment of the present invention, during the PCR process in the micro PCR chip 1, the nucleic acid amplification product (fluorescent substance-bound product) The amplification of the target nucleic acid contained in the initial PCR sample and the reagent and the degree of amplification can be measured and analyzed in real time. In addition, the optical coupler and the optical detector may be disposed on the top or bottom of the column block 200, respectively. However, for the optimal implementation of the real-time PCR apparatus 1000 according to another embodiment of the present invention, the arrangement of the optical coupler and the optical detector may be varied considering the arrangement relationship with other modules, 14 to 15, the light providing portion and the light detecting portion (light detecting module) 300 may be all disposed on the top of the thermal block 200. The light providing unit may include a light emitting diode (LED) light source or a laser light source, a first light filter for selecting light having a predetermined wavelength from the light emitted from the light source, and a second light filter for collecting light emitted from the first light filter And a first aspherical lens including one optical lens and arranged to spread light between the light source and the first optical filter. The light source includes all light sources capable of emitting light, and includes a light emitting diode (LED) light source or a laser light source. The first optical filter selectively emits light of a specific wavelength among incident light having various wavelength ranges and may be variously selected according to the predetermined light source. For example, the first optical filter can pass only light having a wavelength of 500 nm or less among the light emitted from the light source. The first optical lens collects the incident light and increases the intensity of the emitted light. The intensity of the light irradiated to the micro PCR chip 1 through the heat block 200 can be increased. In addition, the light providing unit may further include a first aspherical lens arranged to spread light between the light source and the first optical filter. By adjusting the arrangement direction of the first aspherical lens, the light range emitted from the light source is expanded to reach the measurable area. The optical detecting unit includes a second optical lens for collecting light emitted from the micro PCR chip 1, a second optical filter for selecting light having a predetermined wavelength in light emitted from the second optical lens, And a second aspheric lens disposed between the second optical filter and the optical analyzer for collecting the light emitted from the second optical filter, and a second aspheric lens disposed between the second optical filter and the second optical filter for detecting the optical signal from the light emitted from the second optical filter. A photodiode arranged to remove noise of light emitted from the second aspherical lens and amplify light emitted from the second aspheric lens, between the second aspherical lens and the optical analyzer, an integrated circuit, a PDIC). The second optical lens collects the incident light and increases the intensity of the emitted light. The intensity of the light emitted from the micro PCR chip 1 is increased through the thermal block 200, . The second optical filter selects and emits light of a specific wavelength among incident light having various wavelength ranges. The second optical filter may be selected in various ways according to the wavelength of the predetermined light emitted from the micro PCR chip 1 through the thermal block 200 . For example, the second optical filter can pass only the light of 500 nm or less of the predetermined light emitted from the micro PCR chip 1 through the thermal block 200. The optical analyzer is a module for detecting an optical signal from the light emitted from the second optical filter. The fluorescence analyzer converts the fluorescence emitted from the PCR sample and the reagent into an electric signal to enable qualitative and quantitative measurement. The optical detector may further include a second aspherical lens disposed between the second optical filter and the optical analyzer so as to collect light emitted from the second optical filter. By adjusting the arrangement direction of the second aspherical lens, the optical area emitted from the second optical filter is expanded to reach the measurable area. The photodetector may further include a photodiode arranged to remove noise of light emitted from the second aspherical lens between the second aspherical lens and the optical analyzer and to amplify light emitted from the second aspheric lens, And may further include a photodiode integrated circuit (PDIC). By using the photodiode integrated device 340, it is possible to reduce the size of the device, minimize the noise, and measure the reliable optical signal. In addition, the real-time PCR apparatus 1000 according to another embodiment of the present invention adjusts the traveling direction of light so that the light emitted from the light providing unit can reach the light detecting unit, separates light having a predetermined wavelength Lt; RTI ID = 0.0 > a < / RTI > dichroic filter. The dichroic filter is a module that selectively transmits light or selectively reflects light according to a wavelength. The dichroic filter is disposed at an angle of about 45 degrees with respect to the optical axis of the light emitted from the optical waveguide. The light is selectively transmitted through the short wavelength component according to the wavelength, and the long wavelength component is reflected at a right angle, 200). ≪ / RTI > The dichroic filter is disposed at an angle of about 45 degrees with respect to the optical axis of the light reflected from the micro-PCR chip 1 and the column block 200, and selectively transmits the short wavelength component according to the wavelength And reflects the long wavelength component at a right angle to reach the photodetector portion. The light reaching the photodetector may be converted into an electrical signal by the optical analyzer to indicate amplification or amplification of the nucleic acid.

16 to 18 show a real-time PCR apparatus having two column blocks to which a micro PCR chip according to an embodiment of the present invention is applied.

16 to 18, a real-time PCR apparatus 2000 according to another embodiment of the present invention includes a micro-PCR chip 1 according to an embodiment of the present invention as described above; A first column block 100a disposed on the substrate 400a and configured to be in thermal contact with the micro PCR chip 1; A second column block 200a disposed on the substrate 400a and spaced apart from the first column block 100a and configured to be in thermal contact with the micro PCR chip 1; A chip holder 300a mounted on the first column block 100a and the second column block 200a and capable of moving left and right and / or up and down by the driving means 500a, on which the micro PCR chip 1 is mounted; And the micro-PCR chip (1) is disposed between the first column block (100a) and the second column block (200a), wherein the micro-PCR chip (1) And an optical detection module 700a or 800a configured to detect an optical signal generated from the PCR amplification product inside the PCR reaction chamber 10 of the micro PCR chip 1 when moving between the blocks 200a.

Referring to FIG. 16, a real-time PCR apparatus 2000 according to another embodiment of the present invention includes a first column block 100a disposed on a substrate 400a; A second column block 200a disposed on the substrate 400a and spaced apart from the first column block 100a; And a micro-PCR chip 1 according to an embodiment of the present invention, which is movable left and right and / or up and down by the driving means 500a on the first column block 100a and the second column block 200a, And a mounted chip holder 300a.

The substrate 400a is heated and maintained in temperature by the first and second thermal blocks 100a and 200a so that the physical and / or chemical properties of the first and second thermal blocks 100a, And all materials having a material that prevents mutual heat exchange between the two column blocks 200a. For example, the substrate 400a may include or be made of a material such as plastic.

The first column block 100a and the second column block 200a are for maintaining a temperature for performing a denaturation step, an annealing step, and an extension (or amplification) step for amplifying the nucleic acid. Accordingly, the first column block 100a and the second column block 200a may include or be operably coupled to various modules for providing and maintaining the required temperature required for each of the steps . Therefore, when the chip holder 300a on which the micro PCR chip 1 is mounted is in contact with one surface of each of the thermal blocks 100a and 200a, the first thermal block 100a and the second thermal block 200a, Can heat and maintain the temperature of the contact surface with the micro PCR chip 1 as a whole so that the sample solution in the micro PCR chip 1 can be uniformly heated and maintained at a temperature. Conventionally, a PCR apparatus using a single column block has a temperature change rate in the single column block within a range of 3 to 7 ° C per second, while a real time PCR apparatus including two column blocks according to another embodiment of the present invention 2000), the rate of temperature change in each of the thermal blocks 100a and 200a is in the range of 20 to 40 ° C per second, which can greatly shorten the PCR processing time.

The first column block 100a and the second column block 200a may be provided with heat lines (not shown) therein. The hot wire may be drivably connected to various heat sources to maintain the temperature for performing the denaturation step, the annealing step and the extension (or amplification) step, and may be operably connected to various temperature sensors for monitoring the temperature of the hot wire . In order to keep the internal temperatures of the first column block 100a and the second column block 200a constant, the heat lines are vertically and / or horizontally oriented with respect to the center point of the surfaces of the respective column blocks 100a and 200a And may be arranged to be symmetrical. The arrangement of the hot lines symmetrical in the up and down and / or left and right directions may be varied. In addition, a thin film heater (not shown) may be disposed in the first column block 100a and the second column block 200a. In order to keep the internal temperature of the first column block 100a and the second column block 200a constant, the thin film heater may be vertically and / or horizontally oriented with respect to the center point of the surface of each of the column blocks 100a and 200a Can be spaced apart at regular intervals. The arrangement of the thin film heaters in the vertical direction and / or the horizontal direction may be various.

The first column block 100a and the second column block 200a may include a metal material such as an aluminum material or an aluminum material for uniform heat distribution and rapid heat transfer to the same area.

The first thermal block 100a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) step. For example, the first column block 100a of the real-time PCR device 2000 according to another embodiment of the present invention may maintain 50 ° C to 100 ° C, In the case of carrying out the annealing and extension (or amplification) steps in the first column block 100a, it is possible to maintain a temperature of 55 < 0 > Deg.] C to 75 [deg.] C, and preferably 72 [deg.] C. However, the present invention is not limited thereto, as long as it can perform the above-described denaturation step, or the annealing and extension (or amplification) step. The second thermal block 200a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) step. For example, the second column block 200a of the PCR device according to the third embodiment of the present invention can maintain 90 ° C to 100 ° C when the denaturation step is performed in the second column block 200a, Preferably 95 [deg.] C, and 55 [deg.] C to 75 [deg.] C, preferably 72 [deg.] C, when the annealing and extension (or amplification) steps are performed in the second column block. However, the present invention is not limited thereto, as long as it can perform the above-described denaturation step, or the annealing and extension (or amplification) step. Therefore, the first column block 100a can maintain the denaturation temperature of the PCR. If the denaturation temperature is lower than 90 ° C, the denaturation of the nucleic acid that becomes the template of the PCR occurs, The denaturation step temperature may be 90 ° C to 100 ° C, preferably 95 ° C. When the denaturation step temperature is higher than 100 ° C, the enzyme used for PCR loses its activity. have. In addition, the second column block 200a may maintain the annealing and extension (or amplification) temperature of the PCR. If the extension (or amplification) step temperature is lower than 55 ° C, the specificity of the PCR product may decrease. If the annealing and extension (or amplification) step temperature is higher than 74 ° C, extension by the primer may not occur. The temperature of the annealing and extension (or amplification) step may be 55 ° C to 75 ° C, preferably 72 ° C.

The first column block 100a and the second column block 200a may be spaced apart from each other by a predetermined distance so that mutual heat exchange does not occur. Accordingly, in the nucleic acid amplification reaction which can be significantly influenced even by a minute temperature change since heat exchange does not occur between the first column block 100a and the second column block 200a, Accurate temperature control of the annealing and extension (or amplification) stages is possible.

The real-time PCR apparatus 2000 according to another embodiment of the present invention is capable of moving left and right and / or up and down by the driving means 500a above the first column block 100a and the second column block 200a, And a chip holder 300a on which the PCR chip 1 is mounted. The chip holder 300a is a module in which the micro PCR chip 1 is mounted in the real time PCR apparatus 2000. The inner wall of the chip holder 300a is connected to the micro PCR chip 1 so that the micro PCR chip 1 does not separate from the chip holder 300a when the nucleic acid amplification reaction is performed by the real- And may have a shape or structure to be fixedly attached to the outer wall of the housing. The chip holder 300a is operatively connected to the driving means 500a. In addition, the micro PCR chip 1 may be removably attached to the chip holder 300a.

The driving means 500a may be configured to move the chip holder 300a on which the micro PCR chip 1 is mounted to the first column block 100a and the second column block 200a, Means. The chip holder 300a on which the micro PCR chip 1 is mounted can reciprocate between the first column block 100a and the second column block 200a by moving the driving means 500a in the left- And the chip holder 300a on which the PCR chip 10 is mounted is brought into contact with and separated from the first column block 100a and the second column block 200a by the upward and downward movement of the driving means 500a . The driving means 500a of the real-time PCR apparatus 2000 shown in FIG. 16 includes a rail 510a extending in the left-right direction and a rail 510a arranged in a slidable manner in the left-right direction through the rail 510a, And a movable connection member 520a, and one end of the connection member 520a is disposed on the chip holder. The right and / or left and / or up and down movement of the driving means 500a can be controlled by control means (not shown) arranged to be drivable inside or outside the PCR apparatus, The contact and separation between the chip holder 300a on which the micro PCR chip 1 is mounted and the first column block 100a and the second column block 200a for the annealing and extension (or amplification) .

17 shows each step of the nucleic acid amplification reaction by movement of the chip holder of the real-time PCR apparatus 2000 according to another embodiment of the present invention. The nucleic acid amplification reaction by the real-time PCR apparatus 2000 is performed by the following steps.

First, an oligonucleotide primer, a DNA polymerase, a deoxyribonucleotide triphosphate (dNTP), a deoxyribonucleotide triphosphate (dNTP), or a deoxyribonucleotide triphosphate (dNTP) oligonucleotide having a sequence complementary to a specific base sequence to be amplified, ) And a PCR buffer (PCR buffer), and mounting the PCR chip 10 to the chip holder 300a. Thereafter, or concurrently, the first thermal block 100a is heated and maintained at a temperature for the denaturation step, for example 90 ° C to 100 ° C, and preferably heated and maintained at 95 ° C. The second thermal block 200 is heated and maintained at a temperature for annealing and extending (or amplifying), for example, 55 ° C to 75 ° C, and preferably heated and maintained at 72 ° C . Thereafter, the micro-PCR chip 1 is moved downward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a on which the micro PCR chip 1 is mounted is inserted into the first column The first denaturation step of the PCR is performed by contacting the block 100a (step x). Thereafter, the micro-PCR chip 1 is moved upward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a, on which the micro PCR chip 1 is mounted, The micro PCR unit 1 is separated from the block 100a to terminate the first denaturation step of PCR and control the connecting member 520a of the driving unit 500a to move the micro PCR chip 1 above the second column block 200a (Step y). Thereafter, the micro-PCR chip 1 is moved downward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a, on which the micro-PCR chip 1 is mounted, The first annealing and extension (or amplification) steps of the PCR are performed by contacting the block 100a (step z). Finally, the micro-PCR chip 1 is moved upward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a, on which the micro-PCR chip 1 is mounted, The micro PCR chip 1 is separated from the block 100a to terminate the first annealing and extension (or amplification) step of the PCR and control the connecting member 520a of the driving means 500a to separate the micro- The nucleic acid amplification reaction is performed by repeating the steps x, y, and z after the nucleic acid amplification reaction is carried out above the nucleic acid amplification reaction unit 100a (circulation step).

FIG. 18 shows a step of observing a nucleic acid amplification reaction in real time using a real-time PCR apparatus 2000 according to another embodiment of the present invention. The real-time PCR apparatus 2000 is disposed between the first column block 100a and the second column block 200a and the micro PCR chip 1 is coupled to the first column block 100a by the driving unit 500a. (700a, 800a) configured to detect an optical signal generated from a PCR amplification product in the PCR reaction chamber (10) of the micro PCR chip (1) when moving between the first column block (100a) and the second column block Specifically, a light source 700a and a photodetector 800a. That is, in the real-time PCR apparatus 2000, a light source 700a is disposed between the first column block 100a and the second column block 200a, and the light source 700a is disposed on the chip holder 300a. A light detecting unit 800a for detecting light emitted from the light source 700a or a light detecting unit 800a for detecting light emitted from the light source 700a between the first column block 100a and the second column block 200a, And a light source 700a may be disposed on the chip holder 300a. The light detecting unit 800a is disposed on the driving unit 500a and the driving unit 900a may include a penetrating unit 530a for passing light emitted from the light source 700a.

The arrangement of the light source 700a and the optical detector 800a enables the degree of nucleic acid amplification in the micro PCR chip 1 to be detected in real time during the nucleic acid amplification reaction by the real time PCR device 2000 do. In order to detect the degree of nucleic acid amplification in the micro PCR chip 1, a separate fluorescent material may be added to the sample solution to be introduced into the micro PCR chip 1. The light source 700a is disposed as widely as possible in a spaced space between the first column block 100a and the second column block 200a and is arranged to emit as much light as possible. The light source 700a may be drivingly connected to a lens (not shown) that collects light emitted from the light source 700a and an optical filter (not shown) that filters light of a specific wavelength band.

The step of detecting the amplification degree of the nucleic acid in the micro PCR chip 1 in real time during the nucleic acid amplification reaction by the real time PCR apparatus 2000 is as follows.

After the completion of the first denaturation step of the PCR, the micro-PCR chip 1 is connected to the second column block 200a from above the first column block 100a by controlling the connecting member 520a of the driving means 500a. Or by controlling the connecting member 520a of the driving means 500a after the completion of the first annealing and extension (or amplification) of the PCR to move the micro PCR chip 1 to the second column block The chip holder 300a on which the micro PCR chip 1 is mounted is controlled by the connecting member 520a of the driving unit 500a to move the chip holder 300a above the first column block 200a And then stops on a spaced space between the first column block 100a and the second column block 200a. Thereafter, light is emitted from the light source 700a, and the emitted light passes through the micro PCR chip 1, specifically, the PCR reaction chamber of the micro PCR chip 1, in which case the PCR reaction The optical detector 800a detects an optical signal generated by the amplification of the nucleic acid in the chamber. In this case, the light having passed through the micro-PCR chip 1 of the light-transmitting material passes through the driving unit 500a, specifically, the penetrating unit 530a disposed on the rail 510a, Can reach. Therefore, by monitoring the reaction result by amplification of the nucleic acid (in which the fluorescent material is bound) in the reaction channel during each circulation step of the PCR in real time, the amount of the target nucleic acid contained in the initial reaction sample can be measured in real time Measurement and analysis.

Experimental Example  1. Micro PCR  Manufacture of chips

As shown in FIG. 12, first to third plates 100, 200, and 300 of a flat plate shape were prepared from a plastic material. The first plate 100 was manufactured to a thickness of 0.5 mm and the second plate 200 was formed to have a thickness of 2 mm by integrating 19 PCR reaction chambers 10 in a central circular area. The third plate 300 has a thickness of 2 mm and a circular groove corresponding to the central circular area is formed on the bottom surface of the third plate 300. The third plate 300 has a light transmitting portion 25 protruding toward the inside of the 19 PCR reaction chambers 10, ≪ / RTI > A flexible packing part 50 capable of engaging with the circular groove of the third plate 300 and the light transmission part 50 was manufactured and attached to the lower end surface of the third plate 300. Thereafter, a double-sided adhesive tape was adhered to the upper portion of the first plate 100 and the second plate 200 was attached to the upper portion of the first plate 100. In this case, it is needless to say that the first plate 100 and the second plate 200 can be attached through thermal bonding, ultrasonic bonding, ultraviolet bonding, solvent bonding, etc. in addition to the double-sided adhesive tape. Thereafter, the PCR sample and the reagent are injected into the 19 PCR reaction chambers 10 formed by the attachment of the second plate 200, and the third plate 300 to which the flexible packing part 50 is attached And the PCR reaction chamber 10 was sealed by attaching to the upper portion of the second plate 200. According to Fig. 19, in the present experimental example, the completed micro PCR chip (1) can be identified. FIG. 19A is an external view of the micro PCR chip 1 according to an embodiment of the present invention, FIG. B is an external view of the micro PCR chip 1 of FIG. A reflecting the perspective view of the third plate 300, C is an enlarged view showing a state in which 19 unit modules 50 are arranged in the central circular area of the micro PCR chip 1 of the B figure.

Experimental Example  2. Detection of food poisoning bacteria primer  Set production and synthesis

The primers used for the real-time detection of 16 kinds of food poisoning bacteria were prepared through Primer 3 so that the GC% was set to 40 to 60% and the Tm value was set to 65 to 75 ° C. The prepared primer was commissioned by Genotech Co., Were synthesized. The list of genes specifically amplified in the above primers and each food poisoning bacteria is shown in Table 1 below. In Table 1 below, the primer name refers to the name of the target gene. For example, the target gene in Enteropathogenic E. coli (EPEC) is eaeA gene, the target gene in Enteroinvasive E. coli (EIEC) is invA gene, the gene targeted in Campylobacter coli is ceuE gene, Vibrio The gene targeted in cholerae is ompW gene, and Vibrio The target gene in vulnificus is the Vvh gene.

Strain name Product
you
(bp) Primer
name Sequence Listeria monocytogenes 246 IAP-F CGG ACG TTT AAC CAA GTT GCG CTA AC (SEQ ID NO: 1) IAP-R AGC TGG GAT TGC GGT AAC AGC ATT TG (SEQ ID NO: 2) Staphylococcus aureus 169 NUCA-F AGT TCG TCA AGG CTT GGC TAA AGT TG (SEQ ID NO: 3) NUCA-R GCA GCA GTG ACA CTT TTA CAA TGA GC (SEQ ID NO: 4) Shigella spp .
136 IPAH-F ACC GCC TTT CCG ATA CCG TCT C (SEQ ID NO: 5) IPAH-R CTC TCA GTG GCA TCA GCA GCA ACA G (SEQ ID NO: 6) Vibrio parahaemolyticus 155 TOXR-F CCA AAT AGT AAT TCG CTC GCT GAC CAA CA (SEQ ID NO: 7) TOXR-R AAA CCT TGC TCA CGC CAA ACA AAC TC (SEQ ID NO: 8) Bacillus cereus 157 entFM-F CGG CCG CAC AGG TTA TGT AAG TGC AGA (SEQ ID NO: 9) entFM-R TCC AGC GAT TGA AGA TGT ATC TCC ACC TG (SEQ ID NO: 10) Salmonella spp .
204 INVA-F AAT TAT CGC CAC GTT CGG GCA ATT CGT TA (SEQ ID NO: 11) INVA-R TCA ATA ATA CCG GCC TTC AAA TCG GCA TC (SEQ ID NO: 12) Yersinia enterocolitica 143 AIL-F AAC TGG GGA GTA ATA GGT TCG TTT GCT TA (SEQ ID NO: 13) AIL-R AGG CTA ACA TAT TCG TTG ATG CGG AAA GA (SEQ ID NO: 14) Clostridium perfringens 155 CPE -F ACA ATT TAA ATC CAA TGG TGT TCG AAA ATG  C (SEQ ID NO: 15) CPE -R GGG TTC CCC TAA TAT CCA ACC ATC TCC TTT  A (SEQ ID NO: 16) Campylobacter jejuni 200 HIPO -F GCA CTT CCA TGA CCA CCC CTT CCA  A (SEQ ID NO: 17) HIPO -R CCT GCT GAA GAG GGT TTG GGT GGT GC  (SEQ ID NO: 18) Enterotoxigenic  E. coli ( ETEC ) 107 LT-F AAA ACG TTC CGG AGG TCT TAT GCC CAG A (SEQ ID NO: 19) LT-R CGG TTT GTG TTC CTC TCG CGT GAT CAT A (SEQ ID NO: 20) Enterohemorrhagic  E. coli  ( EHEC ) 159 STX1-F GCA ATT CTG GGA AGC GTG GCA TTA (SEQ ID NO: 21) STX1-R CCC CAG AGT GGA TGA ATC CCA CAA (SEQ ID NO: 22) Enteropathogenic E. coli (EPEC) 238
eaeA-F GGT GGT GTC GCT GGT CAT AC (SEQ ID NO: 23) eaeA-R CCT CTG CCG TTC CAT AAT GTT GT (SEQ ID NO: 24) Enteroinvasive E. coli (EIEC) 216
Inv-F1 CGC TTG ATG GGT GGG GTA TCC TGT CTA (SEQ ID NO: 25) Inv-R1 GTC TGC TCT TGG AGT TGA GTC ACT GTC TGT (SEQ ID NO: 26) Campylobacter coli 234
ceuE-F AGG AAT CAA TGC TGT GGA TGA (SEQ ID NO: 27) ceuE-R CTT GCT AGA TAC CAG TAT TCA GGA T (SEQ ID NO: 28) Vibrio cholerae 249
ompW-F TTG CTG ACA CTA AAC ATC TGC CAC CTA CC (SEQ ID NO: 29) ompW-R ACC ACA CAG AAG CGT TGA GGA ACC A (SEQ ID NO: 30) Vibrio vulnificus 213
Vvh-F CCG CCG CAC TTA CAT TGG TCC ATT CG (SEQ ID NO: 31) Vvh-R CCA CTG ACT TCG CCA CCC ACT TTC G (SEQ ID NO: 32)

Experimental Example  3. PCR  Perform

1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 3 and SEQ ID NO: 4 in the primer set for detecting food poisoning bacteria according to an embodiment of the present invention described in Table 1 using the genomic DNA of the food poisoning bacteria obtained in Experimental Example 1 as a template, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: And SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 were introduced into 16 reaction chambers of the micro PCR chip prepared in Experimental Example 1, respectively. One of the remaining three reaction chambers of the micro PCR chip was loaded with a primer ( IPAH-shigelle ) as a negative control, while the other two were subjected to positive control ( influenza NA was introduced. In carrying out this PCR, the composition of the PCR reaction solution and the PCR reaction conditions are shown in the following Tables 2 and 3, and the PCR apparatus was a conventional PCR apparatus of BIO-RAD.

Furtherance Volume ([mu] l) 2 x Master Mix (final concentration 1 x) 200 Forward primer (10 [mu] M) 40 The reverse primer (10 [mu] M) 40 Template DNA (1 ng / ul) 40 Distilled water (D.W.) 80 Total (total) 400

step Temperature (℃) time Cycles Pre-denaturation 95 1 min One Denaturation 95 10 sec 30 Annealing & Extension 68 10 sec

20 is an electrophoresis image showing the result of PCR performed in Experimental Example 3. Fig. According to FIG. 20, when a micro PCR chip containing each primer set for 16 kinds of food poisoning bacteria according to an embodiment of the present invention is used, it can be confirmed that a number of food poisoning bacteria genes are accurately and rapidly detected.

FIG. 21 is a table showing 16 food poisoning bacteria corresponding to the electrophoresis photographs of FIG. 20. Specifically, NC is a negative control, 2 PC is a positive control, No. 1 is a result according to a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, 2 is a result according to a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, 3 shows the result according to the primer set of SEQ ID NO: 5 and SEQ ID NO: 6, 4 shows the result according to the primer set of SEQ ID NO: 15 and SEQ ID NO: 16, 5 shows the result according to the primer set of SEQ ID NO: 17 and SEQ ID NO: No. 6 results in accordance with the primer set of SEQ ID No. 9 and SEQ ID No. 10, 7 results in accordance with the primer set of SEQ ID No. 13 and SEQ ID No. 14, No. 8 corresponds to the result according to the set of primers of SEQ ID No. 11 and SEQ ID No. 12 SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: The result according to the primer set of SEQ ID NO: 22, the result according to the primer set of SEQ ID NO: 23 and SEQ ID NO: 24, the result according to the primer set of SEQ ID NO: 25 and SEQ ID NO: 26, the SEQ ID NO: As a result of the primer set of SEQ ID NO: 28, SEQ ID NO: 15 results in accordance with the primer set of SEQ ID NO: 29 and SEQ ID NO: 30, and SEQ ID NO: 16 shows different results in the primer set of SEQ ID NO: 31 and SEQ ID NO:

<110> NANOBIOSYS Inc <120> Micro chip for detecting food-borne bacteria, real-time PCR          device comprising the same, and method for detecting food-borne          bacteria using the same <130> WPN12073 <160> 32 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 1 cggacgttta accaagttgc gctaac 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 2 agctgggatt gcggtaacag catttg 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 3 agttcgtcaa ggcttggcta aagttg 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 4 gcagcagtga cacttttaca atgagc 26 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 5 accgcctttc cgataccgtc tc 22 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 6 ctctcagtgg catcagcagc aacag 25 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 7 ccaaatagta attcgctcgc tgaccaaca 29 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 8 aaaccttgct cacgccaaac aaactc 26 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 9 cggccgcaca ggttatgtaa gtgcaga 27 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 10 tccagcgatt gaagatgtat ctccacctg 29 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 11 aattatcgcc acgttcgggc aattcgtta 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 12 tcaataatac cggccttcaa atcggcatc 29 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 13 aactggggag taataggttc gtttgctta 29 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 14 aggctaacat attcgttgat gcggaaaga 29 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 15 acaatttaaa tccaatggtg ttcgaaaatg c 31 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 16 gggttcccct aatatccaac catctccttt a 31 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 17 gcacttccat gaccacccct tccaa 25 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 18 cctgctgaag agggtttggg tggtgc 26 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 19 aaaacgttcc ggaggtctta tgcccaga 28 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 20 cggtttgtgt tcctctcgcg tgatcata 28 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 21 gcaattctgg gaagcgtggc atta 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 22 ccccagagtg gatgaatccc acaa 24 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 23 ggtggtgtcg ctggtcatac 20 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 24 cctctgccgt tccataatgt tgt 23 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 25 cgcttgatgg gtggggtatc ctgtcta 27 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 26 gtctgctctt ggagttgagt cactgtctgt 30 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 27 aggaatcaat gctgtggatg a 21 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 28 cttgctagat accagtattc aggat 25 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 29 ttgctgacac taaacatctg ccacctacc 29 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 30 accacacaga agcgttgagg aacca 25 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 31 ccgccgcact tacattggtc cattcg 26 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artifical sequence <400> 32 ccactgactt cgccacccac tttcg 25

Claims (12)

At least 16 PCR reaction chambers with open top faces; And
A light-transmitting material extending along the optical path and projecting toward the inside of the PCR reaction chamber from a part of the sealing surface contacting the open top face by abutting the open top face of the PCR reaction chamber, A cover having a light transmission portion of the light transmission portion;
, Wherein at least 16 PCR reaction chambers
Listeria made of a primer comprising SEQ ID NO: 1, the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence and SEQ ID NO: 15 or more consecutive nucleotides of the monocytogenes iap a primer set for detecting an invasion associated protein gene;
A primer comprising SEQ ID NO: 3 in the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of a 4 consisting Staphylococcus a primer set for detecting the nucA (nuclease) gene of aureus ;
A primer comprising SEQ ID NO: 6 and primer nucleotide sequence 15 or more consecutive nucleotides of a nucleotide sequence containing at least 15 consecutive nucleotides of the SEQ ID NO: 5 consisting of Shigella spp . A primer set for detecting an ipaH (invasive plasmid antigen gene) gene;
Vibrio consisting of a primer comprising the base sequence of SEQ ID NO: 7, a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 8 and a base sequence at least 15 consecutive nucleotides of the a primer set for detecting the toxR gene of parahaemolyticus ;
A primer comprising SEQ ID NO: 9 of the base sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 10 nucleotide sequence at least 15 consecutive nucleotides of a Bacillus comprising enterotoxin of cereus FM a primer set for detecting an entFM gene;
Salmonella consisting of a primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 12 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 11 spp . A primer set for detecting an invA protein of an invA ;
A primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 14 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 13 consisting of Yersinia a primer set for detecting the ail (attachment invasion locus) gene of enterocolitica ;
Clostridium consisting primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the Primer set for detecting the cpe (C. perfringens enterotoxin) gene of perfringens;
A primer comprising SEQ ID NO: 17 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 18 nucleotide sequence at least 15 consecutive nucleotides of the made Campylobacter a primer set for detecting the hypo gene of jejuni ;
( LT ) of Enterotoxigenic E. coli ( ETEC ) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 19 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 20 a primer set for detecting an enterotoxin gene;
STX1 of Enterohemorrhagic E. coli (EHEC) consisting of a primer comprising SEQ ID NO: 22 and a primer of the nucleotide sequence at least 15 consecutive nucleotides of a nucleotide sequence comprising at least 15 consecutive nucleotides of the SEQ ID NO: 21 (shiga-like a primer set for detecting a toxin-1 gene;
Detecting the eaeA gene of Enteropathogenic E. coli ( EPEC ) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set;
Detecting the invA gene of Enteroinvasive E. coli ( EIEC ) comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 25 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 26 Primer set;
A primer containing the primers and SEQ ID NO: 28 nucleotide sequence at least 15 consecutive nucleotides of the nucleotide sequence containing at least 15 consecutive nucleotides of the SEQ ID NO: 27 consisting Camphylobacter a primer set for detecting the ceuE gene of E. coli ;
Vibrio made of a primer comprising SEQ ID NO: 29 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 30 nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the ompW gene of cholera ; And
Vibrio consisting of a primer comprising the base sequence of SEQ ID NO: 31 primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 32 and a nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the Vvh gene of vulnificus ;
And a primer set selected from the group consisting of: &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; micro-polymerase chain reaction chip. The method according to claim 1,
Wherein the PCR reaction chamber is configured to have a liquid sample capacity of 10 μl or less. 3. The method of claim 2,
Wherein the PCR reaction chamber contains 5 to 8 [mu] l of a liquid sample. The method according to claim 1,
Wherein the light transmitting portion is disposed at the center of the closed surface. The method according to claim 1,
Wherein the light transmitting portion is formed to reach the bottom surface of the PCR reaction chamber or to be spaced upwards from the bottom surface of the PCR reaction chamber. The method according to claim 1,
Wherein the cover further comprises a hole passing through the light transmitting portion and a flexible packing portion contacting the open top face of the PCR reaction chamber to seal the open top face, For micro PCR chip. The method according to claim 1,
A micro PCR chip for detecting food poisoning bacteria, which has a flat plate shape. The method according to claim 1,
A first plate of a flat plate shape;
A second plate disposed on the first plate and having a PCR reaction chamber; And
A third plate disposed on the second plate to seal the open top face of the PCR reaction chamber and serve as a cover having the light transmitting portion;
And a micro-PCR chip for detecting food poisoning bacteria. 9. The method of claim 8,
A hole surrounded by the second plate and the third plate so as to penetrate the light transmitting portion and a flexible packing portion which hits the open top face of the PCR reaction chamber to seal the open top face , A micro-PCR chip for detecting food poisoning bacteria. The method according to claim 1,
Further comprising a heat releasing part for releasing heat generated from the PCR reaction chamber to the outside. A micro PCR chip for detecting food poisoning bacteria according to any one of claims 1 to 10;
At least one thermal block arranged to be in thermal contact with at least one surface of the micro-PCR chip for detecting food poisoning bacteria; And
An optical detecting module configured to detect an optical signal generated from a PCR amplification product inside the PCR reaction chamber of the micro-PCR chip for detecting food poisoning bacteria;
Time PCR device. A micro PCR chip for detecting food poisoning bacteria according to any one of claims 1 to 10;
A first column block disposed on the substrate and configured to be in thermal contact with the micro PCR chip;
A second thermal block disposed on the substrate and spaced apart from the first thermal block and configured to be in thermal contact with the micro PCR chip;
A chip holder mounted on the first column block and the second column block, the chip holder being movable in a lateral direction and / or vertically by a driving means, the micro PCR chip mounted; And
Wherein the micro-PCR chip for detecting food poisoning bacteria is disposed between the first column block and the second column block, and when the micro-PCR chip is moved between the first column block and the second column block by the driving unit, An optical detection module configured to detect an optical signal generated from the PCR amplification product;
Time PCR device.

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