KR101456646B1 - Kit and method for detecting food-borne bacteria - Google Patents

Abstract

The present invention relates to a kit for detecting food poisoning bacteria and a method for simultaneously detecting various kinds of food poisoning bacteria using the same. By using the kit for detecting food poisoning bacteria according to one embodiment, it is possible to efficiently detect various kinds of food poisoning bacteria at the same time.

Description

TECHNICAL FIELD The present invention relates to a kit for detecting food poisoning bacteria and a method for detecting food poisoning bacteria using the kit.

The present invention relates to a kit for detecting food poisoning bacteria and a method for simultaneously detecting various kinds of food poisoning bacteria 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.

One specific example is to provide a kit for the detection of food poisoning bacteria comprising a plurality of types of primer sets for detecting food poisoning bacteria in a PCR chip.

Another embodiment provides a method for simultaneously detecting various kinds of food poisoning bacteria in real time using a kit for detecting food poisoning bacteria.

An aspect includes a first plate; A second plate disposed at an upper portion of the first plate and having at least one through-opening channel; And a third plate disposed on the upper portion of the second plate and having a through-hole inlet formed in one region of each of the at least one through-hole opening channel and a through-hole outlet portion formed in another region of the at least one through- In the at least one through-aperture channel,

(a) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2 or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer set for detecting Listeria monocytogenes consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 32 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 32;

(b) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 4 or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer set for detecting Staphylococcus aureus comprising a primer comprising a nucleotide sequence of SEQ ID NO: 34 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 34;

(c) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 5 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 6 or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: primer containing the nucleotide and SEQ ID NO: consisting of a primer comprising 15 or more of the nucleotide sequence of 36 consecutive nucleotides Shigella boydii , Shigella flexneri or Shigella a primer set for detecting sonnei ;

(d) SEQ ID NO: 7 consisting of the nucleotide sequence primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 8 and the nucleotide sequence primer comprising at least 15 consecutive nucleotides of the Clostridium a primer set for detecting perfringers ;

(e) SEQ ID NO: 9 consisting of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides and SEQ ID NO primer containing a 10 nucleotide sequence at least 15 consecutive nucleotides of the Camphylobacter a primer set for detecting jejuni ;

(f) SEQ ID NO: 11 consisting of a base sequence of 15 or more consecutive nucleotides primer and a primer comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 12 including at least one of Bacillus a primer set for detecting cereus ;

(g) SEQ ID NO: 13 consisting of a primer comprising the base 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 14 Yersinia a primer set for detecting enterocolitica ;

(h) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 15 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 16 or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 37 A primer set for detecting a Salmonella genus bacteria consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 38 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 38;

(i) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 17 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 18 or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer set for detecting Vibrio parahaemolyticus consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 20 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 20;

(j) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 21 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 22, 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 39, a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 39 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer comprising 15 or more consecutive nucleotides or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 41 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 42 To detect toxic E. coli Primer set; And

(k) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 25 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 26, 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 29, a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 29 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: A primer comprising 15 or more consecutive nucleotides, a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 43 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 44, Of the 45 nucleotide sequences, 15 or more nucleotides E. coli O157 consisting of nucleotide primers and SEQ ID NO: 46 base sequence of a primer comprising at least 15 consecutive nucleotides of comprising: at least one primer set selected from the group consisting of a primer set for detecting the respective H7 And a kit for detecting food poisoning bacteria.

The term " primer " refers to a single strand that can act as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerase) Lt; / RTI > oligonucleotide. The suitable length of the primer is typically 15 to 30 nucleotides, depending on various factors, for example, temperature and use of the primer. Short primers may generally require lower temperatures to form a sufficiently stable hybridization complex with the template. The terms " forward primer "and" reverse primer "refer to primers that bind respectively to the 3 'and 5' ends of a constant region of a template amplified by a polymerase chain reaction. The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer set according to one embodiment does not need to have a perfectly complementary sequence to a nucleotide sequence that is a template, and it is interpreted that sufficient complementarity within a range capable of hybridizing to the nucleotide sequence and acting as a primer is sufficient. The design of such a primer can be easily carried out by those skilled in the art with reference to the nucleotide sequence of a polynucleotide to be a template. For example, a primer design program (for example, PRIMER 3, VectorNTI program) have. On the other hand, the primer according to one embodiment is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985). For example, the primer may comprise at least 10 or at least 15 consecutive nucleotides in the nucleotide sequence of any one of SEQ ID NOS: 1 to 30, wherein the primers are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30 Or an oligonucleotide having any one of the base sequences.

According to one embodiment, the at least one primer set comprises a first plate; A second plate disposed at an upper portion of the first plate and having at least one through-opening channel; And a third plate disposed on the upper portion of the second plate and having a through-hole inlet formed in one region of each of the at least one through-hole opening channel and a through-hole outlet portion formed in another region of the at least one through- Is included in the at least one through-aperture channel. For example, if a PCR chip for simultaneously detecting 15 types of primers is prepared, a PCR chip can be fabricated so that a total of 17 through-opening channels are included in the PCR chip, including a positive control group and a negative control group. Meanwhile, according to one embodiment, the PCR chip may be implemented with a light-transmitting material.

According to one embodiment, the first plate and the third plate are made of a material selected from the group consisting of polydimethylsiloxane, cycle olefin copolymer, polymethylmetharcylate, polycarbonate, polypropylene Wherein the second plate is made of a material selected from the group consisting of polypropylene carbonate, polyether sulfone, and polyethylene terephthalate and combinations thereof, and the second plate is made of a material selected from the group consisting of polymethyl methacrylate, polycarbonate, Polyamide, polyethylene, polypropylene, polyphenylene ether, polystyrene, polyoxymethylene, polyetheretherketone, polyetheretherketone, polyetheretherketone, polyetheretherketone, , Polytetrafluoroethylene, polytetrafluoroethylene, Selected from the group consisting of polyvinylchloride, polyvinylidene fluoride, polybutyleneterephthalate, fluorinated ethylenepropylene, perfluoralkoxyalkane, and combinations thereof. Or a thermosetting resin or a thermosetting resin material.

According to one embodiment, the through-hole inlet portion of the third plate is 1.0 mm to 3.0 mm in diameter, the through-hole outlet portion has a diameter of 1.0 mm to 1.5 mm, the third plate has a thickness of 0.1 mm to 2 mm, Wherein the thickness of the second plate is 100 占 퐉 to 200 占 퐉, the width of the through-hole opening channel is 0.5 mm to 3 mm, and the length of the through-hole opening channel is 20 mm to 60 mm. This can be controlled according to the amount of the PCR reaction solution contained in the through-hole opening channel.

According to one embodiment, the Enterotoxigenic bacteria detectable from the kit may be, but are not limited to, intestinal toxic E. coli.

According to one embodiment, the Salmonella spp., Which is detectable from the kit, is Salmonella enteritidis , Salmonella bongori and Salmonella enterica .

According to one embodiment, the kit may further comprise a mixture comprising dATP, dCTP, dGTP and dTTP, a DNA polymerase and a detectable label inside the through-channel. The DNA polymerase may be, for example, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus furiosus (Pfu). ≪ / RTI >

 The term "detectable label" refers to an atom or molecule that specifically detects a molecule containing a label, among molecules of the same type without a label, such detectable labels being, for example, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 660, Alexa Fluor 680, Cy2, Cy3.18, Cy3.5, Cy3.5, Alexa Fluor 430, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59 SYTOX Blue, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO-100, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, 1, YO-PRO-3, YOYO-1, YOYO-3 and thiazo le orange), but the present invention is not limited thereto. In addition, the kit may include a buffer solution in the through-channel. Buffer solutions are compounds that are added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by modulating the pH of the amplification reaction. Such buffer solutions are well known in the art, For example, it may be, but is not limited to, Tris, Tricine, MOPS, or HEPES. In addition, the kit may comprise a dNTP mixture (dATP, dCTP, dGTP, dTTP) and a DNA polymerase joiner.

According to one embodiment, the PCR chip may have two or more through-opening channels, specifically, two or more but not more than seventy-five through-opening channels, but it can be arbitrarily controlled depending on the kind of food poisoning bacteria to be detected. Same as.

In another aspect, there is provided a kit comprising: injecting a subject sample suspected of being a pathogenic bacterium infection into at least one through-flow inlet of the kit to perform real-time PCR; And detecting the presence or absence of food poisoning bacteria in the target sample from the real time PCR result.

Hereinafter, a method of simultaneously detecting two or more food-borne pathogens in real time will be described in detail.

First, the method may include performing a real-time PCR by injecting a sample suspected of being a pathogenic bacterium infection into one or more penetrating opening portions of the kit. According to one embodiment, the real time PCR may be performed in a PCR device comprising a thermal block, a light transmissive column block, or two column blocks, respectively. The PCR device including the light-transmitting thermal block or two thermal blocks is manufactured by the present inventor and can perform real-time PCR, and a detailed description of the device will be given later.

The detection method according to one embodiment can be applied to a sample expected to be infected with food poisoning bacteria. The sample includes, but is not limited to, body fluids such as, for example, cultured cells, blood, saliva, and foods such as meat, dairy products, drinks, DNA of food-borne pathogen can be separately extracted from the sample. However, since the cell membrane of the food-borne pathogen is destroyed due to the high temperature generated during the PCR process and the DNA of food-borne bacteria is exposed to the outside, It can also be used directly in PCR reactions.

According to one embodiment, the real-time PCR reaction can be performed using a real-time PCR apparatus developed by the present inventor. The real-time PCR method is a method for detecting and quantifying fluorescence which appears in real time every PCR cycle by the principle of DNA polymerase and FRET using a device in which a thermal cycler and a spectrophotometer are integrated. This method can distinguish specific amplification products from nonspecific amplification products and can easily obtain the results in an automated manner. In the method for detecting food poisoning bacterium according to one embodiment, the real time PCR reaction can be carried out under ordinary conditions known in the art. For example, initial denaturation is performed at 95 ° C for 10 minutes, Denaturation can be carried out at 95 캜 for 5 seconds, annealing and elongation of the primer at 72 캜 for 20 seconds for a total of 30 times.

Finally, the presence or absence of food poisoning bacteria may be confirmed in the target sample from the real time PCR result.

The sikjungdokgyun presence is a number of cycles from when the curve appears to detect a fluorescent marker labeling a PCR product amplified by the real-time PCR process, PCR amplification products have been a certain amount of amplification C _t Can be confirmed by calculating a value. The C _t The calculation of the value can be performed automatically by the program included in the real-time PCR device.

According to one embodiment, the food poisoning bacterium is Listeria monocytogenes , Staphylococcus aureus , Shigella boyi , Shigella flexneri , Shigella sonnei , Clostridium perfringerns , Camphylobacter jejuni , Bacillus cereus , Yersinia enterocolitica, Salmonella enteritidis , Salmonella bongori , Salmonella enterica , Vibrio parahaemolyticus , Enterotoxigenic bacteria, and E. coli O157: H7.

By using the kit for detecting food poisoning bacteria according to one embodiment, it is possible to efficiently detect various kinds of food poisoning bacteria at the same time.

1 shows a light transmissive column block included in a PCR device according to an embodiment of the present invention.
Figure 2a shows the thermal distribution of the thermal block included in the conventional PCR device.
Figure 2B shows the thermal distribution of the light transmissive thermal block included in the PCR device according to one embodiment of the present invention.
FIG. 2C shows a temperature change with time of the light-transmitting thermal block included in the PCR apparatus according to an embodiment of the present invention.
FIG. 3A shows a light-transmitting thermal block included in a PCR apparatus according to an embodiment of the present invention in which a light absorbing layer is disposed in contact with a lower surface of a substrate, FIG. 3B illustrates a light- FIG. 3C shows a light-transmissive thermal block included in the PCR device according to one embodiment of the present invention. FIG. 3C is a schematic view of a light- Transparent antistatic layer included in the PCR device according to one embodiment of the present invention in which a light reflection preventing layer for contacting the light blocking layer is disposed in contact with the upper portion of the insulating protective layer.
4 shows that a PCR chip is arranged on a light-transmissive column block of a PCR apparatus according to an embodiment of the present invention including a light-providing portion and a light detecting portion.
Figure 5 shows the light providing portion of the PCR device according to one embodiment of the present invention in more detail.
FIG. 6 shows the optical detector of the PCR apparatus according to one embodiment of the present invention in more detail.
Figure 7 shows the optical path by a dichroic filter included in a PCR device according to one embodiment of the present invention.
8 shows a cross section of a light-transmitting PCR chip according to another embodiment of the present invention.
9 shows a cross section of a light-transmitting PCR chip according to another embodiment of the present invention in which a double-sided adhesive or a thermoplastic resin or a thermosetting resin is treated.
Figure 10 shows a PCR device comprising two column blocks according to another embodiment of the present invention.
11 shows each step of nucleic acid amplification reaction by movement of a chip holder of a PCR apparatus including two column blocks according to another embodiment of the present invention.
FIG. 12 shows a step of observing a nucleic acid amplification reaction in real time using a PCR apparatus including two column blocks according to another embodiment of the present invention.
FIG. 13 is a graph showing the results of measurement of the concentration of Staphylococcus aureus , Clostridium perfringerns And E. coli O157: H7 at the same time.
FIG. 14 is a graph showing the results of measurement of Vibrio parahemolyticus , E. coli (ETEC) Plasmid DNA 2 , Yesinia enterocolitica and Listeria monocytogenes were detected at the same time.
FIG. 15 is a graph showing the results of measurement of the amount of bacteria detected by using the kit for detecting food poisoning bacteria according to one embodiment of the present invention. jejuni , Salmonella spp , Bacillus cereus And Shigella spp at the same time.
FIG. 16 shows the result of simultaneous detection of the stx1 gene and the uidA gene of E. coli O157 using a kit for the detection of food poisoning bacteria according to one embodiment of the present invention.
FIG. 17 is a graph showing the results of measurement of Vibrio parahaemolyticus was detected.
FIG. 18 is a graph showing the results of measurement of the concentration of E. coli (ETEC) shows the results of detecting the gene of Plasmid DNA Sth 2.
FIG. 19 is a graph showing the results of a method for detecting food poisoning bacteria according to one embodiment of the present invention using Listeria monocytogenes And Staphylococcus aureus were detected at the same time.
20 is a graph showing the results of measurement of Salmonella spp And Shigella spp were detected at the same time.
21 shows the results of detection of E. coli (ETEC) Plasmid DNA 2 using a kit for the detection of food poisoning bacteria according to one embodiment of the present invention.
FIG. 22 shows the results of detection of E. coli O157 using a kit for the detection of food poisoning bacteria according to one embodiment of the present invention.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the invention is not limited to these embodiments.

1 shows a light-transmissive thermal block 100 included in a PCR device according to an embodiment of the present invention.

A PCR apparatus according to an embodiment of the present invention includes a substrate 10, a heating layer 20 including conductive nanoparticles disposed on the substrate 10, an insulating protective layer 30 disposed on the heating layer, And a light-transmissive thermal block (100) having an electrode (40) connected to the heating layer, wherein the upper surface of the light-transmissive thermal block includes a contact portion (50) of the PCR chip in at least a part of the region.

The substrate 10 may be a light-transmissive plate material, and may be a light-transmissive glass or a light-transmitting plastic material. Although the substrate 10 is shown as a flat plate according to FIG. 1, it may have various shapes such as a semi-cylindrical shape and a hemispherical shape. In addition, the substrate 10 supports the heat generating layer 20.

The heating layer 20 serves as a heat source for the optically transparent thermal block 100 for performing the denaturation step of the PCR, the annealing step, and the extension (or amplification) step. The heating layer 20 is disposed on the substrate 10 and includes conductive nanoparticles (not shown). The conductive nanoparticles may be an oxide semiconductor material or a material to which an impurity selected from the group consisting of In, Sb, Al, Ga, C, and Sn is added to the oxide semiconductor material. In addition, the heating layer 20 may have a loose texture structure in which the conductive nanoparticles are physically necked, and may have a close-packed texture depending on the heat treatment conditions of the manufacturing process And may also be implemented in a complete film state. In addition, since the conductive nanoparticles are dispersed in the solvent, the conductive nanoparticles can be easily laminated on the substrate 10, so that the thickness of the heating layer 20 can be easily controlled by controlling the number of layers . In addition, the conductivity of the heating layer 20 can be easily controlled by adjusting the concentration of the dispersion containing the conductive nanoparticles. An adhesion strengthening layer (not shown) may be formed between the substrate 10 and the heating layer 20 to strongly fix the heating layer 20 to the substrate 10. The adhesion-enhancing layer may be formed of silica or polymer, and may include conductive nanoparticles, and may also perform the same role as the exothermic layer. In addition, the heating layer 20 may be transparent. For example, when the visible light ray has a wavelength of 400 to 700 nm and the heat generating layer including the conductive nanoparticles is formed to have a thickness of 1/4 or less of such a wavelength, for example, about 100 nm or less, .

The insulating protection layer 30 is for protecting the heating layer 20 physically and / or electrically, and may include an insulating material. For example, the insulating material may be selected from the group consisting of dielectric oxides, perylene, nanoparticles, and polymer films. Meanwhile, the insulating protection layer 30 may be transparent.

The electrode 40 is directly or indirectly connected to the heating layer 20 to supply power to the heating layer 20. The electrode 40 may be made of various materials capable of supplying electric power, and may be selected from the group consisting of, for example, a metal material, a conductive epoxy, a conductive paste, a solder, and a conductive film. According to FIG. 1, the electrodes 40 are connected to both sides of the heating layer 20, but can be connected and arranged in various operable positions if power can be supplied to the heating layer 20. In addition, the electrode 40 may be electrically connected to a power source included in the PCR apparatus or disposed externally. For example, the electrode 40 directly contacts the heating layer 20 and connects the heating layer 20 to an external circuit (not shown) through a wiring (not shown) The terminals can be disposed so as to be stably fixed to the electrode 40.

The light-transmissive thermal block 100 includes a chip contact portion 50 in which a PCR chip (not shown) is in contact with at least a portion of the upper surface thereof. The PCR chip may be heated or cooled according to the heat supply or recovery of the light-transmitting heat block 100 by contacting the chip contact unit 50, thereby performing each reaction step of the PCR. In addition, the PCR chip may directly or indirectly contact the chip contact portion 50. [ Meanwhile, the PCR apparatus according to an exemplary embodiment of the present invention may further include modules for performing other PCR including the optically transparent thermal block, and the detailed modules not described herein may include modules It is premised that they are all within the scope.

The PCR device comprising the light-transmissive thermal block 100 has many advantages over conventional PCR devices that use conventional heaters, ceramic heaters or metal heaters as heat blocks. First, since the conductive nanoparticles are used as a heat source, there is no fear of disconnection of the heating layer, and since the conductive nanoparticles are directly heated, high thermal efficiency and low power consumption can be obtained (for example, The heat can be generated at a voltage of about 12V when the heat block is about 2X2 cm), and since it is not made of a metal material, oxidation and corrosion are hardly occurred and durability is excellent. In addition, since light transmittance can be obtained in manufacturing the substrate 10, the heat generating layer 20, and the insulating protective layer 30, it can be included in the sample solution when it is implemented together with the photo- Real-time monitoring of the PCR by the fluorescent material is possible. Since the thickness of the substrate 10, the heat generating layer 20 and the insulating protective layer 30 can be easily controlled, the light transmitting heat block 100 can be made slim, and the light transmitting heat block 100 ) Can be miniaturized. In addition, since the conductive nanoparticles are uniformly distributed in the heating layer 20, uniform heat distribution and rapid temperature control of the light-transmitting thermal block 100 can be performed, the detection efficiency of the PCR result is high, You can get it quickly. The uniformity of the thermal distribution of the light-transmissive thermal block 100 and the rapidity of the temperature control can be confirmed as an experimental result according to FIG. 2A shows the thermal distribution of the thermal block included in the conventional PCR device, FIG. 2B shows the thermal distribution of the light-transmitting thermal block 100 included in the PCR device according to one embodiment of the present invention, and FIG. 2C Illustrates the temperature change over time of the light transmissive thermal block 100 included in the PCR device according to one embodiment of the present invention. In the conventional PCR apparatus, electric power is applied to a cast heater, a ceramic heater or a metal heater used as a thermal block to observe a temperature distribution. In the light-transmitting thermal block 100 according to an embodiment of the present invention, And the temperature distribution was observed. As a result, according to FIG. 2A, the temperature distribution on the conventional heater is not uniform over the heater surface, but according to FIG. 2B, the temperature distribution on the light-transmitting heat block 100 is observed to be uniform as a whole . In addition, power was applied to the light-transmissive thermal block 100 according to an embodiment of the present invention through the electrode 40 to observe the temperature change of the light-transparent heat block 100 over time. As a result, the temperature rise width showed a maximum of 17 ° C / sec, which is a considerably higher value than that of the typical conventional heaters (for example, Bio-Rad's CFX96) .

3A shows a light-transmitting thermal block 100 included in a PCR apparatus according to an embodiment of the present invention in which a light absorbing layer 60 is disposed in contact with a lower surface of a substrate 10, 30 shows a light-transmissive thermal block 100 included in a PCR apparatus according to an embodiment of the present invention in which a light reflection preventing layer 70 is disposed in contact with an upper surface of the substrate 10, A layer 60 is disposed in contact with the insulating layer 30 and a light reflection preventing layer 70 for preventing reflection of light due to contact between the external air layer and the insulating protecting layer 30 is disposed in contact with the upper portion of the insulating protecting layer 30 Transparent thermal block 100 included in a PCR device according to one embodiment of the present invention.

Generally, the presence or absence of the PCR product can be measured and analyzed in real time using the fluorescent material while performing the PCR. Such PCR is referred to as so-called real-time PCR. The reaction involves adding a fluorescent substance as well as reagents necessary for the PCR reaction to the PCR chip, and causing the fluorescent substance to emit light with light of a specific wavelength according to the generation of the PCR product, thereby causing an optical signal that can be measured and analyzed. Therefore, in order to accurately monitor PCR products in real time, it is necessary to increase the sensing efficiency of the optical signal as much as possible. Since the light-transmitting heat block 100 has light transmittance as a whole, most of the excitation light derived from the light source can be transmitted as it is and the sensing efficiency of the optical signal can be increased. However, a portion of the excitation light may be reflected on the optically transmissive thermal block 100 or may be reflected after passing through the optically transmissive thermal block 100 to act as noise in the optical signal. Therefore, preferably, the light absorbing material is treated on the lower surface of the light-transmitting heat block 100 to further enhance the sensing efficiency. 3A, a light absorbing layer 60 is disposed in contact with the lower surface of the substrate 10, and the light absorbing layer 60 includes a light absorbing material. 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 60 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 light reflection preventing material may be treated on the upper surface of the light-transmitting heat block 100 to further enhance the sensing efficiency. 3B, a light reflection preventing layer 70 is disposed in contact with the upper surface of the insulating protection layer 30, and the light reflection preventing layer 70, in combination with the insulating protection layer 30, Prevention function, and includes a light reflection preventing material. 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 it is not limited as long as it is a material capable of preventing reflection of light. Further, more preferably, the light absorbing material is processed on the lower surface of the light-transmitting heat block 100 and the light reflection preventing material is treated on the upper surface of the light-transmitting heat block 100 to further enhance the sensing efficiency . 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, the reflectance of the excitation light of conventional heaters of a common metallic material is about 20 to 80%, but the light intensity of the light according to the present invention including the light absorbing layer 60 or the light reflection preventing layer 70 according to FIG. 3A or FIG. Transmissive column 100 according to the present invention including the light absorbing layer 60 and the light reflection preventing layer 70 according to the present invention can be reduced to 0.2% to 4% When the block 100 is used, the light reflectance can be reduced to 0.2% or less.

4 shows that a PCR chip is arranged on a light-transmissive column block of a PCR apparatus according to an embodiment of the present invention including a light-providing portion and a light detecting portion.

4, the PCR apparatus includes a light supplier 200 disposed to be operable to provide light to a PCR chip 900 disposed on the chip contact unit 50, and a PCR And a light detection unit 300 disposed to be capable of receiving light emitted from the chip 900. [ The optical detector 200 is a module for providing light to the PCR chip 900. The optical detector 300 receives light emitted from the PCR chip 900 and transmits the light to the PCR chip 900 This is a module for measuring the PCR reaction to be performed. Light is emitted from the light supplier 200 and the emitted light passes through or reflects in the reaction chamber (or channel) (not shown) of the PCR chip 900, specifically, the PCR chip 900 In this case, the optical detector 300 detects an optical signal generated by nucleic acid amplification in the reaction chamber (or channel). Accordingly, in the PCR apparatus according to the embodiment of the present invention, in each cycle of the PCR in the PCR chip 900, in the reaction chamber (or channel) By monitoring the reaction result by amplification in real time, it is possible to measure and analyze in real time whether the target nucleic acid contained in the initial sample solution is amplified or amplified. In addition, the optical data providing unit 200 and the optical detecting unit 300 may be disposed on the top or bottom of the optical transmitting column block 900, or may be disposed on the optical block. However, for the optimal implementation of the PCR apparatus according to the present invention, the arrangement of the optical data providing unit 200 and the optical detecting unit 300 may be varied in consideration of the arrangement relationship with other modules, Accordingly, the light providing unit 200 and the light detecting unit 300 may be disposed on the light transmitting block.

FIG. 5 illustrates the optical device 200 of the PCR device according to one embodiment of the present invention in more detail.

5, the light providing unit 200 includes a light emitting diode (LED) light source or a laser light source 210, a first light filter 230 for selecting light having a predetermined wavelength from the light emitted from the light source, And a first optical lens (240) for collecting light emitted from the first optical filter, wherein the first optical filter (230) and the first optical filter (230) And further includes a lens 220. The light source 210 includes all light sources capable of emitting light, and according to an embodiment of the present invention, includes a light emitting diode (LED) light source or a laser light source. The first optical filter 230 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 210. For example, the first optical filter 230 may pass only light having a wavelength of 500 nm or less among the light emitted from the light source 210. The first optical lens 240 collects the incident light and increases the intensity of the emitted light. Thus, the intensity of light irradiated to the PCR chip through the light-transmissive column block 100 can be increased. The optical data providing apparatus 200 may further include a first aspherical lens 220 disposed to spread light between the light source 210 and the first optical filter 230. By adjusting the arrangement direction of the first aspherical lens 220, the light range emitted from the light source 210 is expanded to reach a measurable area.

6 shows the optical detector 300 of the PCR apparatus according to an embodiment of the present invention in more detail.

6, the photodetector 300 includes a second optical lens 310 for collecting light emitted from the PCR chip disposed at the chip contact portion, a second optical lens 310 having a predetermined wavelength in the light emitted from the second optical lens, A second optical filter 320 for selecting light and an optical analyzer 350 for detecting an optical signal from light emitted from the second optical filter, wherein the second optical filter 320 and the optical analyzer And a second aspheric lens 330 disposed between the second aspherical lens 330 and the optical analyzer 350 to collect light emitted from the second optical filter 320 between the second aspherical lens 330 and the optical analyzer 350 A photodiode integrated circuit 340 disposed to remove noise of light emitted from the second aspherical lens 330 and amplify light emitted from the second aspherical lens 330 . The second optical lens 310 collects the incident light and increases the intensity of the emitted light. The intensity of light emitted from the PCR chip is increased through the light-transmitting column block 100, . The second optical filter 320 selectively emits light of a specific wavelength among incident light having various wavelength ranges, and may be variously selected according to the wavelength of the predetermined light emitted from the PCR chip through the light- . For example, the second optical filter 320 may pass only the light having a wavelength of 500 nm or less of the predetermined light emitted from the PCR chip through the optically transparent thermal block 100. The optical analyzer 350 is a module for detecting an optical signal from the light emitted from the second optical filter 320. The optical analyzer 350 converts the light emitted from the sample solution into an electrical signal to enable qualitative and quantitative measurement . The optical detector 300 includes a second aspherical lens 330 disposed between the second optical filter 320 and the optical analyzer 350 to collect light emitted from the second optical filter 320, As shown in FIG. By adjusting the arrangement direction of the second aspherical lens 330, the range of light emitted from the second optical filter 320 is expanded to reach the measurable area. The optical detector 300 may remove noise of light emitted from the second aspherical lens 330 between the second aspherical lens 330 and the optical analyzer 350, And a photodiode integrated circuit (PDIC) 340 arranged to amplify the light emitted from the lens 330. By using the photodiode integrated device 340, it is possible to miniaturize the PCR device, to minimize noise, and to measure a reliable optical signal.

Figure 7 shows the optical path by the dichroic filter 400 included in the PCR device according to one embodiment of the present invention.

Referring to FIG. 7, the PCR apparatus may include a controller for controlling the traveling direction of light so that the light emitted from the light supplier 200 may reach the photodetector unit 300, and for separating light having a predetermined wavelength, And further includes a dichroic filter 400. The dichroic filter 400 is a module that selectively transmits light or selectively reflects light according to a wavelength. 7, the dichroic filter 400a is arranged at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light supplier 200, and selectively transmits the short wavelength component according to the wavelength, To reach the PCR chip 900 disposed on the light-transmissive column block 100. The dichroic filter 400b is disposed at an angle of about 45 degrees with respect to the optical axis of the light reflected from the PCR chip 900 and the light-transmitting column block 100, and selectively arranges the short wavelength component And allows the long wavelength component to be reflected at a right angle to reach the optical detector 300. The light reaching the photodetector 300 is converted into an electrical signal by the optical analyzer to indicate whether the nucleic acid is amplified or amplified.

Fig. 8 shows a cross-section of a light-transmissive PCR chip 900 according to another embodiment of the present invention.

According to Figure 8, a light-transmissive PCR chip 900 is placed on a chip contact 50 included in a light-transmissive thermal block 100 of a PCR device according to one embodiment of the invention described above, And the like. In addition, the light-transmissive PCR chip 900 may be a light-transmitting plastic material. The light-transmitting PCR chip further includes a first plate 910; A second plate (920) disposed on the first plate (910) and having a through-opening channel (921); And a through-hole opening 932 formed in one region of the through-hole opening channel 921 and a through-hole opening outlet 932 formed in another region of the third plate 920, Plate < / RTI >

The PCR chip 900 may include a nucleic acid such as double-stranded DNA, an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, a DNA polymerase, deoxyribonucleotide triphosphates (dNTP), a PCR And a sample solution containing a PCR reaction buffer. The PCR chip 900 includes an inlet 931 for introducing the sample solution, an outlet 932 for discharging the sample solution after completion of the nucleic acid amplification reaction, and an outlet 932 for receiving the sample solution containing the nucleic acid to be amplified (Or channel) 921 as described above. When the PCR chip 900 contacts the optically transparent thermal block 100, the heat of the optically transparent thermal block 100 is transferred to the PCR chip 900 and the PCR reaction chamber of the PCR chip 900 (Or channel) 921 may be heated or cooled to maintain a constant temperature. In addition, the PCR chip 900 may have a planar shape as a whole, but is not limited thereto. In addition, the PCR chip 900 may be indirectly placed in contact with the light-transmitting thermal block 100 while being mounted on a separate chip holder (not shown). Therefore, in one embodiment of the present invention, the fact that the PCR chip 900 is disposed in the chip contact portion 50 of the light-transmitting thermal block 100 means that the PCR chip 900 is mounted on a separate chip holder Transmissive thermal block (100). In addition, the PCR chip 900 may be formed of a light-transmitting material, and preferably includes a light-transmitting plastic material. The PCR chip 900 can increase the heat transfer efficiency only by adjusting the thickness of the plastic using the plastic material, and the manufacturing process can be simplified to reduce the manufacturing cost. In addition, since the PCR chip 900 has a light transmitting property as a whole, it can be directly irradiated to the PCR chip in a state where it is disposed on the chip contact portion 50 of the light-transmitting heat block 100, And the degree of amplification can be measured and analyzed.

The first plate 910 is disposed on the second plate 920. The first plate 910 is adhered to the lower surface of the second plate 920 so that the through-hole opening channel 921 forms a PCR reaction chamber. The first plate 910 may be formed of a variety of materials, but it is preferable to use polydimethylsiloxane, cycle olefin copolymer, polymethylmetharcylate, polycarbonate a material selected from the group consisting of polycarbonate, polypropylene carbonate, polyether sulfone, and polyethylene terephthalate and combinations thereof. In addition, the upper surface of the first plate 910 may be treated with the hydrophilic material 922 to facilitate the PCR. A single layer containing a hydrophilic material 922 may be formed on the first plate 910 by the treatment of the hydrophilic material 922. The hydrophilic material may be various materials, but may be selected from the group consisting of a carboxyl group (-COOH), an amine group (-NH ₂ ), a hydroxyl group (-OH), and a sulfone group (-SH) The treatment of the hydrophilic material can be carried out according to methods known in the art.

The second plate 920 is disposed on the first plate 910. The second plate 920 includes a through-opening channel 921. The through-hole opening channel 921 is connected to a through-opening inlet 931 formed in the third plate 910 and a portion corresponding to the through-opening outlet 932 to form a PCR reaction chamber. Therefore, the sample solution to be amplified is introduced into the through-hole opening channel 921, and the PCR reaction proceeds. In addition, the through-hole opening channel 921 may exist in two or more depending on the purpose and range of use of the PCR apparatus according to one embodiment of the present invention, and six through-opening channels 921 are illustrated according to FIG. 8 . The second plate 920 may be formed of a variety of materials. Preferably, the second plate 920 may be formed of a material such as polymethyl methacrylate, polycarbonate, cycloolefin copolymer, polyamide, polyethylene, polypropylene ), Polyphenylene ether, polystyrene, polyoxymethylene, polyetheretherketone, polytetrafluoroethylene, polyvinylchloride, polyvinylidene chloride, polyvinylidene chloride, A thermoplastic resin or a thermosetting resin selected from the group consisting of polyvinylidene fluoride, polybutyleneterephthalate, fluorinated ethylenepropylene, perfluoralkoxyalkane, and combinations thereof, or a thermosetting resin It can be resin material. Also, the thickness of the second plate 920 may vary, but may be selected from 100 μm to 200 μm. The width and length of the through-hole opening channel 921 may vary, but preferably the width of the through-hole opening channel 921 is selected from 0.5 mm to 3 mm, and the length of the through-hole opening channel 921 May be selected from 20 mm to 60 mm. The inner wall of the second plate 920 may be coated with a material such as silane series or bovine serum albumin in order to prevent DNA and protein adsorption. Can be carried out according to a known method.

The third plate 930 is disposed on the second plate 920. The third plate 930 includes a through-hole inlet 931 formed in one region of the through-hole channel 921 formed in the second plate 920 and a through-hole outlet 932 formed in another region of the third plate 930 do. The penetration opening inlet portion 931 is a portion into which a sample solution containing a nucleic acid to be amplified flows. The through-hole outlet portion 932 is a portion through which the sample solution 932 flows after the completion of the PCR reaction. The third plate 930 covers the through-opening channel 921 formed in the second plate 920 to be described below. The through-hole inlet 931 and the through-hole outlet 932 penetrate through the through- And serves as an inlet portion and an outlet portion of the opening channel 921. The third plate 930 may be made of various materials, but is preferably made of polydimethylsiloxane, cycloolefin copolymer, polymethylmethacrylate, polycarbonate, polypropylene carbonate, polyethersulfone, and polyethylene Terephthalate, terephthalate, and combinations thereof. In addition, the through-hole inlet 931 may have various sizes, but may preferably be selected from 1.0 mm to 3.0 mm in diameter. Further, the through-hole outlet portion 932 may have various sizes, but may preferably be selected from 1.0 mm to 1.5 mm in diameter. The through-hole inlet 931 and the through-hole outlet 932 are provided with separate cover means (not shown). When the PCR reaction for the sample solution proceeds in the through-hole opening channel 921 It is possible to prevent the sample solution from leaking. The cover means may be embodied in various shapes, sizes or materials. In addition, the thickness of the third plate may vary, but may be selected preferably from 0.1 mm to 2.0 mm. In addition, there may be two or more of the through-hole inlet 931 and the through-hole outlet 932.

9 shows a light-transmitting PCR chip according to another embodiment of the present invention in which a double-sided adhesive or a thermoplastic resin or a thermosetting resin 500 is treated. Specifically, the PCR chip according to Fig. 9 can be produced by a method including the following steps.

The light-transmissive PCR chip (100) is mechanically processed to form a through-opening inlet (931) and a through-opening outlet (932) to provide a third plate (930); The plate 930 having a size corresponding to the lower surface of the third plate 930 is inserted into the plate 930 from the portion corresponding to the through-hole inlet 931 of the third plate 930, Forming a through-opening channel 921 through mechanical machining to a portion corresponding to the first plate 932 to provide a second plate 920; Providing a first plate (910) by forming a surface embodied with a hydrophilic material (922) through surface treatment on the upper surface of the plate having a size corresponding to the lower surface of the second plate (920); And the lower surface of the third plate 930 are joined to the upper surface of the second plate 920 through a joining process and the lower surface of the second plate 920 is joined to the upper surface of the first plate 910 And a step of joining to the surface through a bonding step.

The through-opening inlet 931 and the through-hole outlet 932 of the third plate 930 and the through-hole channel 921 of the second plate 920 are formed by injection molding, hot-embossing ), Casting, and laser ablation. ≪ IMAGE > In addition, the hydrophilic material 922 on the surface of the first plate 910 can be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application, . ≪ / RTI > The lower surface of the third plate 930 and the upper surface of the second plate 920 and the lower surface of the second plate 920 and the upper surface of the first plate 910 are thermally bonded, Ultrasonic welding, solvent bonding, or the like, and may be carried out according to methods known in the art. A double-sided adhesive or thermoplastic resin 500 may be applied between the third plate 930 and the second plate 920 and between the second plate 920 and the third plate 910.

Figure 10 shows a PCR device comprising two column blocks according to another embodiment of the present invention.

The PCR device comprising the two column blocks comprises a first column block 1100 disposed on a substrate 1400; A second column block 1200 disposed on the substrate 1400 and spaced apart from the first column block 1100; And a chip holder 1300 mounted on the first column block 1100 and the second column block 1200 and capable of moving left and right and / or up and down by the driving means 1500 and equipped with the PCR chip 900 do.

The PCR device including the two column blocks may complete the first cycle by performing the two steps consisting of the extension step and the annealing and extension (or amplification) steps.

The PCR device including the two column blocks includes a first column block 1100 disposed on a substrate 1400 and a second column block 1100 disposed on the substrate 1400 and spaced apart from the first column block 1100. [ (1200).

The substrate 1400 does not change its physical and / or chemical properties due to heating and temperature maintenance of the first column block 1100 and the second column block 1200, And all materials having a material such that mutual heat exchange does not occur between the two column blocks 1200. For example, the substrate 1400 may comprise or be made of a material such as plastic.

The first column block 1100 and the second column block 1200 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 1100 and the second column block 1200 may include or be operably coupled to various modules for providing and maintaining the required temperature required for each of the steps . Accordingly, when the chip holder 1300 equipped with the PCR chip 900 contacts one surface of each of the thermal blocks 1100 and 1200, the first thermal block 1100 and the second thermal block 1200 The contact surface with the PCR chip 900 can be entirely heated and maintained at a temperature so that the sample solution in the PCR chip 900 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 the PCR apparatus including the two column blocks includes a column block 1100, 1200 ) Is in the range of 20 to 40 ° C per second, which can greatly shorten the PCR reaction time.

In the first column block 1100 and the second column block 1200, heat lines (not shown) may be disposed 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 . The heat lines are vertically and / or horizontally oriented with respect to the center points of the surfaces of the heat blocks 1100 and 1200 so as to maintain the internal temperatures of the first and second heat blocks 1100 and 1200 as a whole. 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 1100 and the second column block 1200. The thin film heater may be vertically and / or horizontally oriented with respect to the center point of each of the heat blocks 1100 and 1200 in order to maintain the internal temperatures of the first and second thermal blocks 1100 and 1200 as uniform as a whole 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 1100 and the second column block 1200 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 column block 1100 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 1100 of the PCR device comprising the two column blocks can maintain 50 ° C to 100 ° C, preferably 90 ° C when the denaturation step is performed in the first column block The temperature can be maintained in the range of 55 to 75 DEG C in the case where the annealing and extension (or amplification) steps are performed in the first column block, Lt; RTI ID = 0.0 > 72 C. < / RTI > 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 1200 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 1200 of the PCR device comprising the two column blocks may maintain 90 ° C to 100 ° C when performing the denaturation step in the second column block, preferably 95 ° C ° C., and in the case where the annealing and extension (or amplification) steps are performed in the second thermal block 1200, the temperature can be maintained at 55 ° C. to 75 ° C., and preferably 72 ° C. can be maintained. 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. Accordingly, the first column block 1100 can maintain the denaturing temperature of the PCR reaction. If the denaturation temperature is lower than 90 ° C, denaturation of the nucleic acid that becomes the template of the PCR reaction occurs, The reaction efficiency may be lowered or the reaction may not occur. If the denaturation step temperature is higher than 100 ° C, the enzyme used in the PCR reaction may lose activity. Therefore, the denaturation step temperature may be 90 ° C to 100 ° C, 95 < 0 > C. Also, the second thermal block 1200 may maintain the annealing and extension (or amplification) temperature of the PCR reaction. If the extension (or amplification) step temperature is lower than 55 ° C, the specificity of the PCR reaction product may be lowered, and if the annealing and extension (or amplification) step temperature is higher than 75 ° C, extension by the primer may not occur The temperature of the annealing and the extension (or amplification) step may be 55 ° C to 75 ° C, preferably 72 ° C, since the PCR reaction efficiency is lowered.

The first column block 1100 and the second column block 1200 may be spaced apart from each other by a predetermined distance such that mutual heat exchange does not occur. Accordingly, in the nucleic acid amplification reaction which can be significantly influenced by a minute temperature change since heat exchange does not occur between the first column block 1100 and the second column block 200, Accurate temperature control of the annealing and extension (or amplification) stages is possible.

The PCR device including the two column blocks can be moved left and right and / or up and down by the driving means 1500 on the first column block 1100 and the second column block 1200, And a chip holder 1300 mounted thereon. In addition, the PCR chip 900 may be configured to receive a sample solution containing a nucleic acid to be amplified, which is in contact with one surface of the first column block 1100 or the second column block 1200 .

The PCR chip 900 has been described with reference to FIGS. When the PCR chip 900 contacts the first column block 1100 and the second column block 1200, the columns of the first column block 1100 and the second column block 1200 are connected to the PCR chip 900, and the sample solution contained in the reaction chamber (or channel) of the PCR chip 900 may be heated and maintained at a temperature. In addition, the PCR chip 900 may have a planar shape as a whole, but is not limited thereto. When the nucleic acid amplification reaction is performed by the PCR device including the two column blocks, the outer wall of the PCR chip 900 is connected to the chip holder 1300 so that the PCR chip 900 does not separate from the chip holder 1300. [ And may have a shape and a structure for being fixedly mounted in the inner space of the housing 1300.

The chip holder 1300 is a module in which the PCR chip 900 is mounted in a PCR apparatus including the two column blocks. The inner wall of the chip holder 1300 is connected to the PCR chip 900 so that the PCR chip 900 does not separate from the chip holder 1300 when the nucleic acid amplification reaction is performed by the PCR device including the two thermal blocks. As shown in FIG. The chip holder 1300 is driveably connected to the driving means 1500. In addition, the PCR chip 900 may be detachable from the chip holder 1300.

The driving means 1500 may include all means for making the chip holder 1300 equipped with the PCR chip 900 movable left and right and / or up and down on the first column block 1100 and the second column block 1200 . The chip holder 1300 on which the PCR chip 900 is mounted can reciprocate between the first column block 1100 and the second column block 1200 by the movement of the driving unit 1500 The chip holder 1300 on which the PCR chip 900 is mounted can be brought into contact with and separated from the first column block 1100 and the second column block 1200 by the upward and downward movement of the driving means 1500 have. The driving unit 1500 of the PCR apparatus including the two column blocks includes a rail 1510 extending in the left and right direction and a slider 1510 slidably moved in the left and right direction through the rail 1510, And a chip holder 1300 is disposed at one end of the connecting member 1520. [ The right and / or left and / or up and down movement of the driving means 1500 can be controlled by control means (not shown) drivably arranged inside or outside the PCR apparatus including the two thermal blocks, Means that the chip holder 1300 on which the PCR chip 900 is mounted for the annealing and extension (or amplification) steps of the denaturation step of the PCR and the chip holder 1300 on which the first column block 1100 and the second column block 1200 Contact and separation can be controlled.

11 shows each step of nucleic acid amplification reaction by movement of a chip holder of a PCR apparatus including two column blocks according to another embodiment of the present invention.

The nucleic acid amplification reaction by the PCR apparatus including the two column blocks is performed by the following steps. First, an oligonucleotide primer, a DNA polymerase, a deoxyribonucleotide triphosphate (dNTP) having a sequence complementary to a specific nucleotide sequence to be amplified, a nucleic acid such as double-stranded DNA, , And a PCR reaction buffer are introduced into the chip holder 1300, and the PCR chip 900 is mounted on the chip holder 1300. Thereafter, or concurrently, the first heat block 1100 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 1200 is heated and maintained at a temperature for annealing and extension (or amplification) steps, for example, 55 ° C to 75 ° C, and preferably heated and maintained at 72 ° C .

The PCR chip 900 is moved downward by controlling the connecting member 1520 of the driving unit 1500 so that the chip holder 1300 on which the PCR chip 900 is mounted is inserted into the first column block 1100) to perform the first denaturation step of the PCR (step x).

Thereafter, the PCR chip 900 is moved upward by controlling the connecting member 1520 of the driving unit 1500 so that the chip holder 1300, on which the PCR chip 900 is mounted, 1100 to terminate the first denaturing step of the PCR and control the connecting member 1520 of the driving unit 1500 to move the PCR chip 900 above the second column block 1200 (Step y).

Thereafter, the PCR chip 900 is moved downward by controlling the connecting member 1520 of the driving unit 1500 so that the chip holder 1300, on which the PCR chip 900 is mounted, 1100) to perform a first annealing and extension (or amplification) step of the PCR (step z).

Lastly, the PCR chip 900 is moved upward by controlling the connecting member 1520 of the driving unit 1500 so that the chip holder 1300, on which the PCR chip 900 is mounted, The PCR chip 900 is separated from the first column block 1100 by controlling the connecting member 1520 of the driving unit 1500. The first annealing and extension And repeating the above steps x, y, and z to perform a nucleic acid amplification reaction (circulation step).

FIG. 12 shows a step of observing a nucleic acid amplification reaction in real time using a PCR apparatus including two column blocks according to another embodiment of the present invention.

The PCR apparatus including the two column blocks may further include a light source 1700 disposed between the first column block 1100 and the second column block 1200 and the light source 1700 may be disposed on the chip holder 1300. [ Or a light for detecting light emitted from the light source 1700 between the first column block 1100 and the second column block 1200 may be further disposed, A detection unit 1800 may be further disposed, and a light source 1700 may be further disposed on the chip holder 1300. The light detecting unit 1800 is disposed on the driving unit 1500 and the driving unit 1500 may include a penetrating unit 1530 for passing light emitted from the light source 1700. [

By the arrangement of the light source 1700 and the optical detector 1800, the extent to which the nucleic acid is amplified in the PCR chip 900 is detected in real time during the nucleic acid amplification reaction by the PCR apparatus including the two column blocks . In order to detect the amplification degree of the nucleic acid in the PCR chip 900, a separate fluorescent material may be further added to the sample solution to be introduced into the PCR chip 900. The light source 1700 is arranged to be as widely distributed as possible in the spaced space between the first column block 1100 and the second column block 1200 and is arranged to emit as much light as possible. The light source 1700 may be operably connected to a lens (not shown) for collecting light emitted from the light source 1700 and an optical filter (not shown) for filtering light of a specific wavelength band.

The step of detecting the amplification degree of the nucleic acid in the PCR chip 900 in real time during the nucleic acid amplification reaction by the PCR device including the two column blocks is performed in the following steps. After the completion of the first denaturation step of the PCR, the connection member 1520 of the driving unit 1500 is controlled to move the PCR chip 900 from the top of the first column block 1100 to the top of the second column block 1200 Or controlling the connecting member 1520 of the driving means 1500 after the first annealing and extension (or amplification) step of the PCR to control the PCR chip 900 in the second column block 1200 The chip holder 1300 on which the PCR chip 900 is mounted is controlled by the connecting member 1520 of the driving unit 1500 to move the chip holder 1300 on the first column block 1100, And then stops on a spaced-apart space between the column block 1100 and the second column block 1200. Thereafter, light is emitted from the light source 1700 and the emitted light passes through the reaction chamber (or channel) of the optically transparent PCR chip 900, specifically, the PCR chip 900. In this case, The optical detection unit 1800 detects an optical signal generated by amplification of a nucleic acid in the reaction chamber (or channel). In this case, the light that has passed through the optically transparent PCR chip 900 can pass through the driving unit 1500, specifically, the penetration unit 1530 disposed on the rail 1510 and reach the light output unit 1800 have.

Therefore, according to the PCR apparatus including the two column blocks, the reaction result by amplification of the nucleic acid (the fluorescent substance is bound) in the reaction chamber (or channel) during each circulation step of the PCR reaction is By monitoring in real time, the amount of target nucleic acid contained in the initial reaction sample can be measured and analyzed in real time.

Example  1: Food poisoning culture and genome DNA Extraction of

11 pathogenic microorganisms causing food poisoning were purchased from the Korea Microorganism Conservation Center, Korea BioResource Center and The global bioresource Center. The pathogenic microorganisms were cultured according to the culture conditions shown in Table 1 below, and genomic DNA was extracted using a QiaAmp DNA purification kit (QIAGEN).

badge Strain BHI (Brain Heart infusion): 37
Bacillus Clustridium (Clostridium) (43 anaerobic) Listeria genus (Listeria) Yersinia Shigella LB (Luria Bertani): 37 Salmonella genus (Salmonella) Poisonous Bacterial Genus (ETEC) Vibrio Staphylococcus (Staphylococcus) E. coli CB (Campy BAP), NB (Nutrient Broth) Camphylobacter (42 aerobic)

Example  2: Detection of food poisoning bacteria primer  Production and synthesis

The primers used for the real-time detection of 11 species of food poisoning bacteria were prepared through Primer 3 so that the GC% was 40 to 60%, the Tm value was 65 to 75 ° C, and the primer was commissioned by Genotech Co., Were synthesized. The list of genes specifically amplified in the above primers and in each food poisoning bacteria is shown in Table 2 below. In the following table, the primer name refers to the name of the target gene. For example, the target gene in Staphylococcus aureus is the nuc A gene.

Strain Name Product  size ( bp ) Primer name Sequnce Listeria monocytogenes 221 NBS-IAP-F GCGCCACTACGGACGTTTAACCAAG (SEQ ID NO: 1) NBS-IAP-R ACAATCGCATCCGCAAGCACTGTAG (SEQ ID NO: 2) 246 NBS (N) -IAP-F CGGACGTTTAACCAAGTTGCGCTAAC (SEQ ID NO: 31) NBS (N) -IAP-R AGCTGGGATTGCGGTAACAGCATTTG (SEQ ID NO: 32) Staphylococcus aureus 198 NBS-NUCA-F ATTGGTTGATACACCTGAAACAAAGCATCC (SEQ ID NO: 3) NBS-NUCA-R AAAGCTTCGTTTACCATTTTTCCATCAGCA (SEQ ID NO: 4) 169 NBS (N) -NUCA-F AGTTCGTCAAGGCTTGGCTAAAGTTG (SEQ ID NO: 33) NBS (N) -NUCA-R GCAGCAGTGACACTTTTACAATGAGC (SEQ ID NO: 34) Shigella boyish 153 NBS-IPAH-F ATAATGATACCGGCGCTCTGCTCTCC (SEQ ID NO: 5) Shigella flexneri NBS-IPAH-R AGTTTCTCTGCGAGCATGGTCTGGAA (SEQ ID NO: 6) Shigella sonnei 240 NBS (N) -IPAH-F AACAGGTCGCTGCATGGCTGGAAAAA (SEQ ID NO: 35) NBS (N) -IPH-R TTTATCCCGGGCAATGTCCTCCAGAA (SEQ ID NO: 36) Clostridium perfringerns 155 NBS-CPE-F ACAATTTAAATCCAATGGTGTTCGAAAATGC (SEQ ID NO: 7) NBS-CPE-R GGGTTCCCCTAATATCCAACCATCTCCTTTA (SEQ ID NO: 8) Camphylobacter jejuni 210 NBS-HIPO-F ACCAAAAGGCATATTGTGCCATCCAAA (SEQ ID NO: 9) NBS-HIPO-R GTAATGCATGCTTGTGGTCATGATGGA (SEQ ID NO: 10) Bacillus cereus 187 NBS-entFM-F GTACAAAATTAACCATAACGGCCGCACAG (SEQ ID NO: 11) NBS-entFM-R AGATCTAGCGAATCCAGCGATTGAAGATG (SEQ ID NO: 12) Yersinia enterocolitica 223 NBS-AIL-F GCCATCTTTCCGCATCAACGAATATG (SEQ ID NO: 13) NBS-AIL-R ACCAAGCATCCAGGTGCCAACTTTTA (SEQ ID NO: 14) Salmonella typhimurium 217 NBS-INVA-F GCGCCGCCAAACCTAAAACCA (SEQ ID NO: 15) Salmonella choleraesuis NBS-INVA-R GCAGGCGCACGCCATAATCAA (SEQ ID NO: 16) Salmonella enteritidis 218 NBS (N) -INVA-F TATGTGTTGCGGAACGCGCTTGATGA (SEQ ID NO: 37) Salmonella bongori NBS (N) -INVA-R CGCACGGAAACACGTTCGCTTAACAA (SEQ ID NO: 38) Salmonella enterica Vibrio parahaemolyticus 162 NBS-GYRB-F AAGCGTTCGTTCAGCACTTAAACACCAA (SEQ ID NO: 17) NBS-GYRB-R GCTGTGGAATGTTGTTGGTGAAACAGAA (SEQ ID NO: 18) 224 NBS-TOXR-F ACATCGGCACCAAATTTCTACTTGCTCA (SEQ ID NO: 19) NBS-TOXR-R AGTCAGGCTTGAGTCATCCACCTCAAAA (SEQ ID NO: 20) E. coli (ETEC) Plasmid DNA 2 187 NBS-LT-F GCGTATACAGCCCTCACCCATATGAACA (SEQ ID NO: 21) NBS-LT-R AATCTGTAACCATCCTCTGCCGGAGCTA (SEQ ID NO: 22) 277 NBS (N) -LT-F AAAACGTTCCGGAGGTCTTATGCCCAGA (SEQ ID NO: 39) NBS (N) -LT-R TGGGTGAGGGCTGTATACGCCTAATACA (SEQ ID NO: 40) 233 NBS-STh-F GATGCCATGTCCGGAGGTAATATGAAGAA (SEQ ID NO: 23) NBS-STh-R AATAGCACCCGGTACAAGCAGGATTACAA (SEQ ID NO: 24) 240 NBS (N) -STh-F TCCGTGAAACAACATGACGGGAGGTA (SEQ ID NO: 41) NBS (N) -STh-R CATGGAGCACAGGCAGGATTACAACA (SEQ ID NO: 42) E. coli O157: H7 159 NBS-STX1-F GCAATTCTGGGAAGCGTGGCATTA (SEQ ID NO: 25) NBS-STX1-R CCCCAGAGTGGATGAATCCCACAA (SEQ ID NO: 26) 148 NBS (N) -STX1-F GCGACGCCTGATTGTGTAACTGGAAA (SEQ ID NO: 43) NBS (N) -STX1-R TCATCCCCGTAATTTGCGCACTGAGA (SEQ ID NO: 44) 136 NBS-STX2-F ATGTGGCCGGGTTCGTTAATACGG (SEQ ID NO: 27) NBS-STX2-R GCTGCGACACGTTGCAGAGTGGTA (SEQ ID NO: 28) 225 NBS (N) -STX2-F TTCGCGCCGTGAATGAAGAGAGTCAA (SEQ ID NO: 45) NBS (N) -STX2-R CAATCCGCCGCCATTGCATTAACAGA (SEQ ID NO: 46) 159 NBS-UIDA-F GAATTGAGCAGCGTTGGTGGGAAA (SEQ ID NO: 29) NBS-UIDA-R GGCCTGCCCAACCTTTCGGTATAA (SEQ ID NO: 30)

PCR was performed using the genomic DNAs of the strains obtained in Example 1 as a template in order to confirm the specific detection of the primers for each pathogenic bacterium. The following Table 3 and Table 4 show the composition of the PCR reaction solution used in the PCR reaction and the PCR conditions performed. Each PCR reaction solution was added distilled water to the following composition so that the total volume was 50 μl.

Item density 10X PCR buffer (Kosomogin Tec) 1X 10 mM dNTPs 1 mM Template (genomic DNA) 25 ng Primer (forward / reverse) 1 uM Taq Polymerase (Cosmogyn Tech) 1

Reaction temperature Reaction time 95 (denaturation) 10 seconds 72 (annealing & extension) 10 seconds

(2 steps, 30 cycles)

After completion of the PCR, 3 μl of each reaction solution was subjected to electrophoresis on 1% agarose gel to confirm the PCR reaction product, whereby the specific gene present in each pathogenic bacterium was detected by the primer sets of Table 2 And the remaining 47 μl was electrophoresed on 1% agarose gel. Then, a DNA fragment of the PCR reaction product was extracted using a gel extraction kit (Invitrogen). Each of the extracted DNA fragments was cloned into a StrataClone PCR cloning kit (Stratagene). The cloned vector was transformed into E. coli and then plated on LB plate containing ampicillin / X-gal / IPTG and cultured at 37 ° C. Then, only the colonies containing the cloned vector so as to contain the PCR amplification product were selected and cultured in LB medium. Then, the plasmid DNA was extracted using plasmid DNA miniprep kit (Qiagen).

Example  3: Light transmittance Heat block  Or two Heat block  Each containing PCR  Devices and Light transmittance PCR  Simultaneous Detection of Foodborne Pathogens Using Chip

Using a genomic DNA extracted in Example 1 as a template and using a primer set capable of detecting the 11 types of food poisoning bacteria, a Real-time PCR instrument (a light-transmitting thermal block or two column blocks manufactured by the present inventor And a light-permeable PCR chip. The composition of the reaction solution for real-time PCR and the reaction conditions for real-time PCR are shown in Tables 5 and 6 below. Each PCR reaction solution was added with distilled water to a total volume of 12 μl in the following composition, and the reaction solution for detecting different kinds of food poisoning bacteria was injected into the through-hole inlet of different through-channel opening in the PCR chip. In the present embodiment, six through-opening channels are arranged on the PCR chip so that six types of food poisoning bacteria can be detected at the same time. As a positive control, the plasmid pSC-HA containing the HA (Hemagglutinin) gene of New Influenza A virus H1N1 was used as a template instead of the genomic DNA.

Item density 10X PCR buffer 1X 10 mM dNTPs 1 mM Template (genomic DNA) 25 ng Primer (forward / reverse) 1 uM Taq polymerase 3U SYBR Green Dye 1X

Reaction temperature Reaction time 95 (denaturation) 5 seconds 72 (annealing & extension) 14 seconds

(2 steps, 30 cycles)

As shown in FIG. 13 to FIG. 22, it was confirmed that each of the food-borne pathogens can be detected in real time by using the kit according to one embodiment of the present invention.

<110> NANOBIOSYS INC. <120> Kit and method for detecting food-borne bacteria <130> PN089984 <160> 46 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-IAP-F) <400> 1 gcgccactac ggacgtttaa ccaag 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer (NBS-IAP-R) <400> 2 acaatcgcat ccgcaagcac tgtag 25 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-NUCA-F) <400> 3 attggttgat acacctgaaa caaagcatcc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-NUCA-R) <400> 4 aaagcttcgt ttaccatttt tccatcagca 30 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer (NBS-IPAH-F) <400> 5 ataatgatac cggcgctctg ctctcc 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer (NBS-IPAH-R) <400> 6 agtttctctg cgagcatggt ctggaa 26 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-CPE-F) <400> 7 acaatttaaa tccaatggtg ttcgaaaatg c 31 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-CPE-R) <400> 8 gggttcccct aatatccaac catctccttt a 31 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-HIPO-F) <400> 9 accaaaaggc atattgtgcc atccaaa 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-HIPO-R) <400> 10 gtaatgcatg cttgtggtca tgatgga 27 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-entFM-F) <400> 11 gtacaaaatt aaccataacg gccgcacag 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-entFM-R) <400> 12 agatctagcg aatccagcga ttgaagatg 29 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-AIL-F) <400> 13 gccatctttc cgcatcaacg aatatg 26 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-AIL-R) <400> 14 accaagcatc caggtgccaa ctttta 26 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-INVA-F) <400> 15 gcgccgccaa acctaaaacca 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-INVA-R) <400> 16 gcaggcgcac gccataatca a 21 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-GYRB-F) <400> 17 aagcgttcgt tcagcactta aacaccaa 28 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer (NBS-GYRB-R) <400> 18 gctgtggaat gttgttggtg aaacagaa 28 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-TOXR-F) <400> 19 acatcggcac caaatttcta cttgctca 28 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer (NBS-TOXR-R) <400> 20 agtcaggctt gagtcatcca cctcaaaa 28 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-LT-F) <400> 21 gcgtatacaga ccctcaccca tatgaaca 28 <210> 22 <211> 28 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-LT-R) <400> 22 aatctgtaac catcctctgc cggagcta 28 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-STh-F) <400> 23 gatgccatgt ccggaggtaa tatgaagaa 29 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-STh-R) <400> 24 aatagcaccc ggtacaagca ggattacaa 29 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-STX1-F) <400> 25 gcaattctgg gaagcgtggc atta 24 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-STX1-R) <400> 26 ccccagagtg gatgaatccc acaa 24 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-STX2-F) <400> 27 atgtggccgg gttcgttaat acgg 24 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer (NBS-STX2-R) <400> 28 gctgcgacac gttgcagagt ggta 24 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS-UIDA-F) <400> 29 gaattgagca gcgttggtgg gaaa 24 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS-UIDA-R) <400> 30 ggcctgccca acctttcggt ataa 24 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > forward primer (NBS (N) -IAP-F) <400> 31 cggacgttta accaagttgc gctaac 26 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -IAP-R) <400> 32 agctgggatt gcggtaacag catttg 26 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS (N) -NUCA-F) <400> 33 agttcgtcaa ggcttggcta aagttg 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -NUCA-R) <400> 34 gcagcagtga cacttttaca atgagc 26 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS (N) -IPAH-F) <400> 35 aacaggtcgc tgcatggctg gaaaaa 26 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -IPAH-R) <400> 36 tttatcccgg gcaatgtcct ccagaa 26 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > forward primer (NBS (N) -INVA-F) <400> 37 tatgtgttgc ggaacgcgct tgatga 26 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -INVA-R) <400> 38 cgcacggaaa cacgttcgct taacaa 26 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer (NBS (N) -LT-F) <400> 39 aaaacgttcc ggaggtctta tgcccaga 28 <210> 40 <211> 28 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -LT-R) <400> 40 tgggtgaggg ctgtatacgc ctaataca 28 <210> 41 <211> 26 <212> DNA <213> Artificial Sequence <220> The forward primer (NBS (N) -STh-F) <400> 41 tccgtgaaac aacatgacgg gaggta 26 <210> 42 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -STh-R) <400> 42 catggagcac aggcaggatt acaaca 26 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > forward primer (NBS (N) -STX1-F) <400> 43 gcgacgcctg attgtgtaac tggaaa 26 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -STX1-R) <400> 44 tcatccccgt aatttgcgca ctgaga 26 <210> 45 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > forward primer (NBS (N) -STX2-F) <400> 45 ttcgcgccgt gaatgaagag agtcaa 26 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> Reverse primer (NBS (N) -STX2-R) <400> 46 caatccgccg ccattgcatt aacaga 26

Claims (11)

A PCR device for detecting food poisoning bacteria,
A first plate, a second plate disposed on the upper portion of the first plate, the second plate having at least two through-aperture channels, and a second plate disposed at an upper portion of the second plate, A third plate having a through-hole opening formed therein and a through-opening opening formed in another region;
A first column block disposed on the substrate;
A second column block disposed on the substrate and spaced apart from the first column block; And
And a chip holder mounted on the first column block and the second column block and being movable left and right and up and down by driving means,
Wherein the first column block and the second column block maintain different temperatures from each other,
The PCR is performed by making the reciprocal contact between the first column block and the second column block by the movement of the chip holder,
Each of the through-hole channels includes a set of primers for detecting different pathogenic bacteria,
The primer set includes:
(a) a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2; Or a primer set for detecting Listeria monocytogenes consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 31 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 32;
(b) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 4; Or a primer set for detecting Staphylococcus aureus consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 33 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 34;
(c) a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 5 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 6; Or a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 35 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 36, Primer set;
(d) a primer set for detecting Clostridium perfringerns consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 8;
(e) a primer set for detecting Camphylobacter jejuni consisting of a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 9 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 10;
(f) a primer set for detecting Bacillus cereus consisting of a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 11 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 12;
(g) a primer set for detecting Yersinia enterocolitica consisting of a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 14;
(h) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 15 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 16; Or a primer set for detecting a Salmonella genus bacteria consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 37 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 38;
(i) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 17 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 18; Or a primer set for detecting Vibrio parahaemolyticus consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 19 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 20;
(j) a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 21 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 22; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 39 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 40; Or a primer set for detecting intestinal toxic E. coli comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 41 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 42; And
(k) a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 25 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 26; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 27 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 28; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 29 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 30; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 43 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 44; Or a primer set for detecting E. coli O157: H7 consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 45 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 46 &Lt; / RTI &gt; The PCR device of claim 1, wherein the PCR chip is implemented with a light-transmitting material. The method of claim 1, wherein the first and third plates are formed from a material selected from the group consisting of polydimethylsiloxane, cycle olefin copolymer, polymethylmetharcylate, polycarbonate, Wherein the second plate is made of a material selected from the group consisting of polypropylene carbonate, polyether sulfone, and polyethylene terephthalate and combinations thereof, and the second plate is made of a material selected from the group consisting of polymethyl methacrylate, polycarbonate, Polyamide, polyethylene, polypropylene, polyphenylene ether, polystyrene, polyoxymethylene, polyetheretherketone, polyetheretherketone, polyetheretherketone, polyetheretherketone, , Polytetrafluoroethylene, poly Wherein the polymer is selected from the group consisting of polyvinylchloride, polyvinylidene fluoride, polybutyleneterephthalate, fluorinated ethylenepropylene, perfluoralkoxyalkane, and combinations thereof. A thermoplastic resin or a thermosetting resin. The apparatus of claim 1, wherein the through-opening inlet of the third plate has a diameter of 1.0 mm to 3.0 mm, the through-hole outlet has a diameter of 1.0 mm to 1.5 mm, the third plate has a thickness of 0.1 mm to 2 mm, Wherein the thickness of the second plate is 100 占 퐉 to 200 占 퐉, the width of the through-hole opening channel is 0.5 mm to 3 mm, and the length of the through-hole opening channel is 20 mm to 60 mm. The PCR device according to claim 1, wherein the Salmonella bacteria are selected from the group consisting of Salmonella enteritidis , Salmonella bongori, and Salmonella enterica . 2. The PCR device of claim 1, wherein the PCR device further comprises a mixture comprising dATP, dCTP, dGTP and dTTP, a DNA polymerase and a detectable label inside the through-channel. 7. The method of claim 6 wherein the detectable label is selected from the group consisting of Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, , Alexa Fluor 680, Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, , SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, Wherein the PCR product is selected from the group consisting of SEQ ID NO: 2, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 and thiazole orange. delete Performing a real-time PCR by injecting a target sample suspected of being a pathogenic bacterium infection into at least two through-hole inflow portions of the PCR apparatus of any one of claims 1 to 7; And
And detecting the presence or absence of food poisoning bacteria in the target sample from the real time PCR result. delete 10. The method according to claim 9, wherein the food poisoning bacterium is Listeria monocytogenes , Staphylococcus aureus, Shigella boyi , Shigella flexneri , Shigella sonnei , Clostridium perfringerns, Camphylobacter jejuni , Bacillus cereus , Yersinia enterocolitica , Salmonella enteritidis , Salmonella bongori , Salmonella enterica , Vibrio parahaemolyticus , intestinal toxic E. coli, and E. coli O157: H7.

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