KR101483493B1 - PCR device for detecting food-borne bacteria, and and method for detecting food-borne bacteria using the same - Google Patents

Abstract

The present invention relates to a primer set for detecting food poisoning, a PCR apparatus using the same, and a method for detecting food poisoning using the same. According to the present invention, it is possible to accurately and promptly confirm the infection with food poisoning bacteria at low cost, It can contribute greatly to take.

Description

Technical Field [0001] The present invention relates to a primer set for detecting food poisoning, a PCR apparatus using the same, and a method for detecting food poisoning using the same,

The present invention relates to a primer set for the detection of food poisoning bacteria, a PCR chip containing the same, a PCR apparatus containing the PCR chip, 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.

An embodiment of the present invention provides a primer set for detecting food poisoning bacteria capable of detecting at least one of 16 food poisoning bacteria.

Another embodiment of the present invention is to provide a PCR chip for detecting food poisoning bacteria comprising a primer set for detecting food poisoning bacteria capable of detecting at least one of five food poisoning bacteria in a PCR chip, and a PCR apparatus including the same.

In addition, another embodiment of the present invention provides a method for detecting various kinds of food poisoning bacteria simultaneously and in real time using a PCR chip for detecting food poisoning bacteria and a PCR apparatus including the same.

First embodiment of the present invention is a Listeria monocytogenes comprising primers comprising the primers and SEQ ID NO: 2 of the nucleotide sequence at least 15 consecutive nucleotides of comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 1 iap ( a primer set for detecting an invasion associated protein gene; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer for detecting the nucA (nuclease) gene of Staphylococcus aureus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: set; A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 6 . A primer set for detecting an ipaH (invasive plasmid antigen gene) gene; A primer set for detecting a toxR gene of Vibrio parahaemolyticus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 8; A primer comprising SEQ ID NO: 9 of the base sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 10 nucleotide sequence at least 15 consecutive nucleotides of a Bacillus comprising enterotoxin of cereus FM a primer set for detecting an entFM gene; Salmonella consisting of a primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 12 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 11 spp . A primer set for detecting an invA protein of an invA ; A primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 14 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 13 consisting of Yersinia a primer set for detecting the ail (attachment invasion locus) gene of enterocolitica ; Clostridium consisting primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the Primer set for detecting the cpe (C. perfringens enterotoxin) gene of perfringens; A primer comprising SEQ ID NO: 17 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 18 nucleotide sequence at least 15 consecutive nucleotides of the made Campylobacter a primer set for detecting the hypo gene of jejuni ; ( LT ) of Enterotoxigenic E. coli ( ETEC ) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 19 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 20 a primer set for detecting an enterotoxin gene; STX1 of Enterohemorrhagic E. coli (EHEC) consisting of a primer comprising SEQ ID NO: 22 and a primer of the nucleotide sequence at least 15 consecutive nucleotides of a nucleotide sequence comprising at least 15 consecutive nucleotides of the SEQ ID NO: 21 (shiga-like a primer set for detecting a toxin-1 gene; Detecting the eaeA gene of Enteropathogenic E. coli ( EPEC ) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set; Detecting the invA gene of Enteroinvasive E. coli ( EIEC ) comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 25 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 26 Primer set; A primer containing the primers and SEQ ID NO: 28 nucleotide sequence at least 15 consecutive nucleotides of the nucleotide sequence containing at least 15 consecutive nucleotides of the SEQ ID NO: 27 consisting Camphylobacter a primer set for detecting the ceuE gene of E. coli ; Vibrio made of a primer comprising SEQ ID NO: 29 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 30 nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the ompW gene of cholera ; Vibrio consisting of a primer comprising the base sequence of SEQ ID NO: 31 primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 32 and a nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the Vvh gene of vulnificus ; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 33 and a STX2 (shiga-like ) gene of Enterohemorrhagic E. coli ( EHEC ) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: a primer set for detecting a toxin-2 gene; And SEQ ID NO: 35 base sequence of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 36 and the nucleotide sequence of Enterotoxigenic E. coli (ETEC) consisting of 15 or more consecutive nucleotides of a primer containing ST (heat- stable enterotoxin gene), wherein the primer set is selected from the group consisting of primers set for detection of stable enterotoxin gene.

A second embodiment of the present invention relates to a first plate, A second plate disposed on the first plate and having at least one reaction channel; And a third plate disposed on the second plate and having an inlet and an outlet connected to both ends of the at least one reaction channel and configured to be openable and closable, And a PCR (Polymerase Chain Reaction) chip comprising the primer set according to the first embodiment. In a second embodiment of the present invention, the first and third plates are made of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmetharcylate (PMMA) ), Polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof. And the second plate comprises a material selected from the group consisting of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) ), Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate a thermoplastic resin or a thermosetting resin material selected from the group consisting of polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof. In addition, the PCR chip may be formed of a plastic material, and may be implemented to have optical transparency. In addition, the PCR chip may further comprise a mixture comprising dATP, dCTP, dGTP, and dTTP, a DNA polymerase and a detectable label in the at least one reaction channel. In addition, the detectable label may be 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, , Cy3, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Blue, SYTOX Green, SYTOX Green, SYTOX Green, , SYTOX Orange, SYBR Green, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3 and thiazole orange.

A third embodiment of the present invention provides a semiconductor device comprising: a first column block disposed on a substrate; A second column block disposed on the substrate and spaced apart from the first column block; And a chip holder mounted on the first column block and the second column block, the chip holder being movable left and right and / or up and down by a driving means and having a PCR chip according to the second embodiment of the present invention. In the third embodiment of the present invention, a light source is further disposed between the first column block and the second column block, and a light detecting portion for detecting light emitted from the light source is further disposed on the chip holder, A light detector is further disposed between the first column block and the second column block for detecting light emitted from the light source, and a light source may further be disposed on the chip holder.

A fourth embodiment of the present invention is directed to a liquid crystal display comprising a substrate, a heating layer including conductive nanoparticles disposed on the substrate, an insulating protective layer disposed on the heating layer, and an electrode connected to the heating layer, A light-transmissive thermal block embodied to have a plurality of light- And a PCR chip according to the second embodiment of the present invention disposed so as to be able to contact the upper surface of the light-transmitting thermal block. In the fourth embodiment of the present invention, the substrate is made of a light-transmitting glass or a plastic material, and the conductive nanoparticles included in the heating layer are formed of an oxide semiconductor material or an oxide semiconductor material of In, Sb, Al, Ga, Sn, and the insulating protective layer is selected from the group consisting of a dielectric oxide, a perylene, a nanoparticle and a polymer film, and the electrode is made of a metal material, a conductive epoxy, a conductive paste, And a conductive film. In addition, a light absorbing layer containing a light absorbing material may be disposed in contact with the lower surface of the substrate of the light transmitting block, or a light reflecting layer may be formed on the upper surface of the insulating protective layer of the light transmitting block, Can be disposed in contact with each other. In addition, the PCR device may further include an optical detector disposed to be operable to provide light to the PCR chip disposed on the chip contact portion, and an optical detector disposed movably to receive light emitted from the PCR chip disposed on the chip contact portion. As shown in FIG.

In a fifth embodiment of the present invention, there is provided a heater group including at least one heater, at least two heater groups, and at least two heater groups are repeatedly arranged in such a manner that two or more heater units are spaced apart from each other, A thermal block having a contact surface of a PCR chip on at least one side of which a target sample is received; An electrode unit having an electrode connected to supply electric power to the heaters provided in the column block; And a PCR chip according to the second embodiment of the present invention, which is disposed so as to be able to make heat exchange with one or more heaters provided in the thermal block, on the thermal block. In a fifth embodiment of the present invention, the thermal block may include two to four heater groups. The thermal block may include two heaters, the first heater group may maintain the PCR denaturation temperature, the second heater group may maintain the PCR annealing / extension temperature, The PCR annealing / extension step temperature can be maintained and the second heater group can maintain the PCR denaturation step temperature. The heat block includes three heater groups, the first heater group maintains the PCR denaturation temperature, the second heater group maintains the PCR annealing temperature, and the third heater group maintains the PCR extension temperature , Or the first heater group maintains the PCR annealing step temperature, the second heater group maintains the PCR extension step temperature, the third heater group maintains the PCR denaturation step temperature, or the first heater group Group can maintain the PCR extension step temperature, the second heater group can maintain the PCR denaturation step temperature, and the third heater group can maintain the PCR annealing step temperature. Also, the thermal block may be implemented to have optical transparency. In addition, the heater provided in the thermal block may include a light-transmitting heat generating element. The apparatus may further include a power supply for supplying power to the electrode unit and a pump arranged to provide a positive pressure or a negative pressure to control a flow rate and a flow rate of the fluid flowing in the at least one reaction channel. Further, a light source is disposed between the first heater and the second heater, and a power supply for supplying electric power to the electrode unit; a positive pressure or negative pressure control unit for controlling a flow rate and a flow rate of the fluid flowing in the at least one reaction channel; A pump arranged to provide negative pressure, and a light detecting section for detecting light emitted from the light source. A pump arranged to provide a positive or negative pressure to control the flow rate and flow rate of fluid flowing in the at least one reaction channel; a pump arranged to supply light to the PCR chip; And an optical detector disposed to receive the light emitted from the PCR chip.

The sixth embodiment of the present invention comprises the steps of: introducing a sample suspected of causing foodborne pathogen infection into the at least one reaction channel of the PCR chip according to the second embodiment of the present invention; And checking the presence or absence of food poisoning bacteria in the target sample from the PCR result.

In the sixth embodiment of the present invention, the PCR is performed using the PCR apparatus according to the third embodiment of the present invention, the PCR apparatus according to the fourth embodiment of the present invention, and the PCR according to the fifth embodiment of the present invention Lt; RTI ID = 0.0 > PCR < / RTI >

According to one embodiment of the present invention, a primer set capable of specifically amplifying a gene relating to food poisoning bacteria, a PCR chip including the same, a PCR device including the PCR chip, and a method for detecting food poisoning bacteria using the same, It can be confirmed accurately and promptly at cost, which can greatly contribute to prevent the spread of food poisoning bacteria and to take prompt response measures.

1 is a diagram showing a PCR chip according to a second embodiment of the present invention.
2 is a view showing a PCR chip according to a second embodiment of the present invention in which a double-sided adhesive or a thermoplastic resin or a thermosetting resin is treated.
3A to 3C are views showing a PCR apparatus according to a third embodiment of the present invention.
4A to 4I are views showing a PCR apparatus according to a fourth embodiment of the present invention.
5A to 5H are views of a PCR apparatus according to a fifth embodiment of the present invention.
6A, 6B, 6C, and 6B show primer sets of SEQ ID NOs: 1 and 2, primer sets of SEQ ID NOs: 3 and 4, primer sets of SEQ ID NOs: 5 and 6, primer sets of SEQ ID NOs: 7 and 8, 13 and 14, and a primer set of SEQ ID NOs: 15 and 16, a primer set for detecting a food poisoning bacterium according to an embodiment of the present invention, a third embodiment of the present invention 6B is an electrophoresis image of a primer set of SEQ ID NOs: 17 and 18, a primer set of SEQ ID NOs: 19 and 20, a primer set of SEQ ID NOs: 21 and 22 The primer set of SEQ ID NOs: 23 and 24, the primer set of SEQ ID NOs: 25 and 26, the primer set of SEQ ID NOs: 27 and 28, the primer set of SEQ ID NOs: 29 and 30, Real-time PCR results for samples using a PCR apparatus according to the third embodiment of the present invention on the premise of a primer set for detecting food poisoning bacteria according to an embodiment of the present invention comprising primer sets Nos. 31 and 32 It is an electrophoresis photograph.
7A, 7B and 8, a primer set of SEQ ID NOs: 9 and 10, a primer set of SEQ ID NOs: 3 and 4, a primer set of SEQ ID NOs: 13 and 14, and a primer set of SEQ ID NOs: 15 and 16, a primer set for detecting a food poisoning bacterium according to an embodiment of the present invention, a third embodiment of the present invention 7B is a graph showing the results of real-time PCR for a sample using a PCR apparatus according to the example. Fig. 7B is a graph showing the results of a real-time PCR using the primer set of SEQ ID NOs: 17 and 18, the primer set of SEQ ID NOs: 19 and 20, The primer set of SEQ ID NOs: 23 and 24, the primer set of SEQ ID NOs: 25 and 26, the primer set of SEQ ID NOs: 27 and 28, the primer set of SEQ ID NOs: 29 and 30, And a primer set of 32 according to an embodiment of the present invention is a graph showing real-time PCR results of a sample using a PCR apparatus according to the third embodiment of the present invention on the assumption of a primer set for detecting food poisoning bacteria .
FIGS. 8A and 8B show Ct value values according to the real-time PCR results of FIGS. 7A and 7B.
9A is a graph showing the results of a real-time PCR using a PCR apparatus according to the third embodiment of the present invention, assuming a primer set for detecting food poisoning bacteria according to an embodiment of the present invention comprising the primer sets of SEQ ID NOS: 33 and 34, FIG. 9B is a photograph showing the result of PCR performed on the basis of the primer set for detecting food poisoning bacteria according to one embodiment of the present invention, which is composed of the primer set of SEQ ID NOS: 35 and 36, and the PCR apparatus according to the third embodiment of the present invention The results of the electrophoresis are shown in Fig.
FIG. 10A is a graph showing the results of a real-time PCR using a PCR apparatus according to the third embodiment of the present invention on the premise of a primer set for detecting food poisoning bacteria according to an embodiment of the present invention comprising primer sets of SEQ ID NOs: FIG. 10B is a graph showing the results of the PCR, and FIG. 10B is a graph showing the results obtained by using the PCR apparatus according to the third embodiment of the present invention, assuming that the primer set for detecting food poisoning bacteria is composed of the primer set of SEQ ID NOs: 35 and 36 FIG. 2 is a graph showing real-time PCR results for a sample.

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

The primer set for detecting food poisoning bacteria according to the first embodiment of the present invention comprises 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: Listeria monocytogenes iap a primer set for detecting an invasion associated protein gene; A primer comprising SEQ ID NO: 3 in the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of a 4 consisting Staphylococcus a primer set for detecting the nucA (nuclease) gene of aureus ; Shigella spp consisting of a primer comprising the base sequence of SEQ ID NO: 5 primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 6 and a nucleotide sequence at least 15 consecutive nucleotides of the. A primer set for detecting an ipaH (invasive plasmid antigen gene) gene; Vibrio consisting of a primer comprising the base sequence of SEQ ID NO: 7, a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 8 and a base sequence at least 15 consecutive nucleotides of the a primer set for detecting the toxR gene of parahaemolyticus ; A primer comprising SEQ ID NO: 9 of the base sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 10 nucleotide sequence at least 15 consecutive nucleotides of a Bacillus comprising enterotoxin FM of cereus a primer set for detecting an entFM gene; Salmonella consisting of a primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 12 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 11 spp . A primer set for detecting an invA protein of an invA ; A primer comprising a primer and a nucleotide sequence SEQ ID NO: 15 or more consecutive nucleotides of the 14 comprising a nucleotide sequence at least 15 consecutive nucleotides of the SEQ ID NO: 13 consisting of Yersinia a primer set for detecting the ail (attachment invasion locus) gene of enterocolitica ; Detecting the cpe (C. perfringens enterotoxin) gene of Clostridium perfringerns made of a primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the A primer set; A primer comprising SEQ ID NO: 17 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 18 nucleotide sequence at least 15 consecutive nucleotides of the made Camphylobacter a primer set for detecting the hypo gene of jejuni ; ( LT ) of Enterotoxigenic E. coli ( ETEC ) 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 a primer set for detecting an enterotoxin gene; STX1 of Enterohemorrhagic E. coli (EHEC) consisting of a primer comprising SEQ ID NO: 22 and a primer of the nucleotide sequence at least 15 consecutive nucleotides of a nucleotide sequence comprising at least 15 consecutive nucleotides of the SEQ ID NO: 21 (shiga-like a primer set for detecting a toxin-1 gene; Detecting the eaeA gene of Enteropathogenic E. coli ( EPEC ) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set; Detecting the invA gene of Enteroinvasive E. coli ( EIEC ) comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 25 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 26 Primer set; A primer containing the primers and SEQ ID NO: 28 nucleotide sequence at least 15 consecutive nucleotides of the nucleotide sequence containing at least 15 consecutive nucleotides of the SEQ ID NO: 27 consisting Camphylobacter a primer set for detecting the ceuE gene of E. coli ; Vibrio made of a primer comprising SEQ ID NO: 29 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 30 nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the ompW gene of cholera ; Vibrio consisting of a primer comprising the base sequence of SEQ ID NO: 31 primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 32 and a nucleotide sequence at least 15 consecutive nucleotides of the a primer set for detecting the Vvh gene of vulnificus ; A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 33 and a STX2 (shiga-like ) gene of Enterohemorrhagic E. coli ( EHEC ) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: a primer set for detecting a toxin-2 gene; And SEQ ID NO: 35 base sequence of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 36 and the nucleotide sequence of Enterotoxigenic E. coli (ETEC) consisting of 15 or more consecutive nucleotides of a primer containing ST (heat- stable enterotoxin) gene.

The primer may be a single strand of oligonucleotides capable of acting as a starting point for template-directed DNA synthesis under appropriate conditions (i.e., four different nucleoside triphosphates and polymerase) . 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. In this case, a forward primer and a reverse primer refer to a primer that binds to the 3 'end and the 5' end of a constant region of a template amplified by a polymerase chain reaction, respectively. The sequence of the primer does not need to have a sequence completely complementary to the 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 the first embodiment of the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence which is a template, and it suffices to have sufficient complementarity within a range capable of hybridizing to the sequence to perform the primer action . 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. Meanwhile, the primer according to one embodiment of the present invention 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 primers are identical to those of SEQ ID NO: 1 and SEQ ID NO: 2 (SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: (SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 4 and SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10).

A method for detecting food poisoning bacteria using the above primer set is as follows. First, PCR is performed by introducing a sample suspected of having a pathogenic bacterium infection into the above-mentioned one or more reaction channels of a PCR chip to be described in detail below. Target sample refers to a sample (sample or sample solution) of an individual suspected of being infected with food poisoning bacteria, including, but not limited to, cultured cells, blood, saliva, and the like. In this case, a sample suspected of being a pathogenic bacterium infection may include a step of extracting DNA from the sample. Thereafter, the extracted DNA is introduced into a PCR chip and real-time PCR is performed. Therefore, the presence or absence of food poisoning bacteria can be confirmed from the PCR result. In this case, the PCR may be performed in a series of PCR apparatuses to be described in detail below. Further, when a real-time PCR apparatus is used, PCR is performed using a PCR amplification product (Ct value) which is the number of cycles when the product is amplified by a certain amount. It is a matter of course that the calculation of the Ct value can be automatically performed by a program installed in the real-time PCR apparatus.

Referring to FIG. 1, a PCR chip according to a second embodiment of the present invention includes a first plate 11; A second plate (12) disposed on the first plate (11) and having at least one reaction channel (14); And a third plate (12) disposed on the second plate (12) and having an inlet (15) and an outlet (16) connected to both ends of the at least one reaction channel (14) 13), wherein the at least one reaction channel (14) comprises a primer set according to the first embodiment of the present invention.

In addition to the primer set according to the first embodiment of the present invention, deoxyribonucleotide triphosphates (dNTP), specifically dATP, dCTP, dGTP, and dTTP , A DNA polymerase, a detectable label, and a PCR buffer (PCR buffer). 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 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 660, , Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43 SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, SYTOX Green, YOYO-1, YOYO-3, and thiazole orange. The present invention also relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. The PCR buffer is a compound added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction. Such buffers are known, and include, for example, Tris, Tricine, MOPS, or HEPES. The PCR chip 10 comprises an inlet 15 for introducing the sample solution, an outlet 16 for discharging the sample solution after completion of the nucleic acid amplification reaction, and an outlet 15 for receiving the sample solution containing the nucleic acid to be amplified. Or more of the reaction channel 14. When the PCR chip 10 contacts the thermal block of the PCR device, the heat generated in the thermal block is transferred to the PCR chip 10, and the sample included in the reaction channel 14 of the PCR chip 10 The solution may be heated or cooled to maintain a constant temperature. In addition, the PCR chip 10 may have a planar shape as a whole, but is not limited thereto. In addition, the PCR chip 10 may be placed in contact with the thermal block while being mounted on a chip holder of the PCR device. Accordingly, the fact that the PCR chip 10 is disposed on one side of the thermal block includes that the PCR chip 10 is disposed in contact with the thermal block in a state where the PCR chip 10 is mounted on the chip holder. In addition, the PCR chip 10 may be formed of a plastic material, and may have a light transmitting property. The PCR chip 10 can increase the heat transfer efficiency only by adjusting the thickness of the plastic using the plastic material, and the manufacturing process can be simplified, thereby reducing the manufacturing cost. In addition, since the PCR chip 10 can have light transmittance as a whole, it can be directly irradiated with light in a state where it is disposed on one side of the thermal block, and the amplification degree and amplification degree of nucleic acid can be measured and analyzed in real time .

The first plate (11) is disposed on the lower surface of the second plate (12). The first plate 11 is adhered to the lower surface of the second plate 920 so that the at least one reaction channel 14 forms a PCR reaction chamber. The first plate 11 may be formed of a variety of materials, but preferably includes polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the like. In addition, the upper surface of the first plate 11 may be treated with the hydrophilic material 17 to smoothly perform the PCR. A single layer comprising a hydrophilic material 17 may be formed on the first plate 11 by the treatment of the hydrophilic material 17. The hydrophilic material may be a variety of materials but is preferably selected from the group consisting of a carboxyl group (-COOH), an amine group (-NH2), a hydroxyl group (-OH), and a sulfone group (-SH) The treatment of the hydrophilic material can be carried out according to methods known in the art.

The second plate 12 is disposed on the upper surface of the first plate 11. The second plate (920) includes at least one reaction channel (14). The reaction channel 14 is connected to a portion corresponding to the inflow portion 15 and the outflow portion 16 formed in the third plate 13 to form a PCR reaction chamber. Therefore, after the sample solution to be amplified is introduced into the reaction channel 921, the PCR proceeds. In addition, the reaction channel 14 may exist in two or more depending on the purpose and range of use of the PCR apparatus, and according to FIG. 1, six reaction channels 14 are illustrated. The second plate 12 may be made of various materials, but preferably includes polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC) Polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetherimide (POM) , Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate , PBT), fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof. It is chosen or a thermoplastic resin may be a thermosetting resin material. The thickness of the second plate 12 may vary, but may be selected from 100 탆 to 200 탆. The width and length of the reaction channel 14 may vary but preferably the width of the reaction channel 14 is selected from 0.5 mm to 3 mm and the length of the through channel 14 is 20 mm to 40 mm. The inner wall of the second plate 12 may be coated with a material such as a silane series or bovine serum albumin (BSA) to prevent adsorption of DNA or protein, Can be carried out according to methods known in the art.

The third plate (13) is disposed on the second plate (12). The third plate 13 has an inlet 15 formed in one region on one or more reaction channels 921 formed in the second plate 12 and an outlet 16 formed in another region. The inlet 15 is a portion into which a sample solution containing a nucleic acid to be amplified flows. The outflow portion 16 is a portion through which the sample solution flows out after the completion of the PCR. The third plate 13 covers at least one reaction channel 14 formed in the second plate 12 which will be described below and the inlet 15 and the outlet 16 are connected to the reaction channel 14 As shown in FIG. The third plate 13 may be made of various materials, but it is preferable to use polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the like. In addition, the inlet 15 may have various sizes, but may preferably be selected from 1.0 mm to 3.0 mm in diameter. In addition, the outlet 16 may have various sizes, but may preferably be selected from 1.0 mm to 1.5 mm in diameter. The inflow section 15 and the outflow section 16 are provided with separate cover means (not shown) to prevent the sample solution from leaking when the PCR for the sample solution proceeds in the reaction channel 14 . 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, two or more of the inflow section 15 and the outflow section 16 may exist.

According to FIG. 2, the PCR chip 10 may be formed by mechanical processing to form an inlet 15 and an outlet 16 to provide a third plate 13; A plate having a size corresponding to the lower surface of the third plate 13 is attached to the outflow portion 16 of the third plate 13 from a portion corresponding to the inflow portion 15 of the third plate 13 Forming at least one reaction channel (14) through mechanical processing to a corresponding portion to provide a second plate (12); Providing a first plate (11) by forming a surface embodied with a hydrophilic material (17) on the upper surface of the plate material having a size corresponding to the lower surface of the second plate (12) through surface treatment; And the lower surface of the third plate 13 is joined to the upper surface of the second plate 12 through a joining process and the lower surface of the second plate 12 is joined to the upper surface of the first plate 11 And a step of joining to the surface through a bonding step. The inlet 15 and outflow 16 of the third plate 13 and the reaction channel 14 of the second plate 12 are formed by injection molding, hot-embossing, casting ), And laser ablation. ≪ IMAGE > In addition, the hydrophilic material 17 on the surface of the first plate 11 may be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application, . ≪ / RTI > The lower surface of the third plate 13 and the upper surface of the second plate 12 and the lower surface of the second plate 12 and the upper surface of the first plate 11 are thermally bonded, Ultrasonic welding, solvent bonding, and may be performed according to methods known in the art. A double-sided adhesive or a thermoplastic resin or a thermosetting resin 18 may be applied between the third plate 13 and the second plate 12 and between the second plate 12 and the third plate 13.

Hereinafter, a PCR apparatus in which the PCR chip according to the second embodiment of the present invention is driven will be described. PCR apparatus is a device for use in PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific nucleotide sequence. For example, a PCR apparatus for amplifying a deoxyribonucleic acid (DNA) having a specific nucleotide sequence can be produced by heating a sample solution containing double stranded DNA at a specific temperature, for example, about 95 ° C, A denaturing step of separating the DNA into stranded DNA and an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified in the sample solution, Annealing step of annealing the sample solution after the annealing step to form a partial DNA-primer complex by binding the primer to a specific base sequence of the single strand DNA by cooling to, for example, 55 ° C, The primers of the partial DNA-primer complexes are prepared on the basis of the DNA polymerase by maintaining the temperature at, for example, 72 < DNA having the specific nucleotide sequence can be amplified exponentially by performing an extension step (or amplification step) for forming double stranded DNA and repeating the above three steps, for example, 20 to 40 cycles. have. In some cases, the PCR device may simultaneously perform the annealing step and the extension (or amplification) step, in which case the PCR device may perform the two steps consisting of the extension step and the annealing and extension (or amplification) step , Thereby completing the first cycle. Accordingly, in this specification, a PCR apparatus refers to a device including modules for performing the above steps, and the detailed modules not described herein are all included in the scope of the prior art for performing PCR, It is assumed that it is doing.

3A to 3C, a PCR apparatus according to a third embodiment of the present invention will be described in detail.

3A, a PCR apparatus according to a third embodiment of the present invention includes a first column block 100a disposed on a substrate 400a; A second column block 200a disposed on the substrate 400a and spaced apart from the first column block 100a; And the PCR chip 10 according to the second embodiment of the present invention is capable of moving left and right and / or up and down by the driving means 500a on the first column block 100a and the second column block 200a, And a mounted chip holder 300a.

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

The first column block 100a and the second column block 200a are for maintaining a temperature for performing a denaturation step, an annealing step, and an extension (or amplification) step for amplifying the nucleic acid. Accordingly, the first column block 100a and the second column block 200a may include or be operably coupled to various modules for providing and maintaining the required temperature required for each of the steps . Accordingly, when the chip holder 300a on which the PCR chip 10 is mounted is in contact with one surface of each of the thermal blocks 100a and 200a, the first thermal block 100a and the second thermal block 200a The contact surface with the PCR chip 10 can be entirely heated and maintained at a temperature so that the sample solution in the PCR chip 10 can be uniformly heated and maintained at a temperature. Conventionally, in a PCR apparatus using a single column block, the rate of temperature change in the single column block is within a range of 3 to 7 ° C per second, whereas the PCR apparatus including two column blocks according to the third embodiment of the present invention The rate of temperature change in each of the thermal blocks 100a and 200a is in the range of 20 to 40 ° C per second, which can greatly shorten the PCR processing time.

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

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

The first thermal block 100a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) step. For example, the first column block 100a of the PCR device according to the third embodiment of the present invention may maintain 50 ° C to 100 ° C, and preferably the denaturation step in the first column block 100a The annealing and extension (or amplification) steps in the first column block 100a may be performed at a temperature of 55 ° C to 75 ° C, 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 200a may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) step. For example, the second column block 200a of the PCR device according to the third embodiment of the present invention can maintain 90 ° C to 100 ° C when the denaturation step is performed in the second column block 200a, Preferably 95 [deg.] C, and 55 [deg.] C to 75 [deg.] C, preferably 72 [deg.] C, when the annealing and extension (or amplification) steps are performed in the second column block. However, the present invention is not limited thereto, as long as it can perform the above-described denaturation step, or the annealing and extension (or amplification) step. Therefore, according to the third embodiment of the present invention, the first column block 100a can maintain the denaturing temperature of the PCR, and when the denaturation temperature is lower than 90 ° C, The efficiency of the PCR may be lowered or the reaction may not occur. If the temperature of the denaturation step is higher than 100 ° C, the enzyme used for PCR loses its activity. Therefore, the denaturation step temperature is 90 ° C to 100 ° C Lt; RTI ID = 0.0 > 95 C. < / RTI > In addition, according to the third embodiment of the present invention, the second column block 200a can maintain the annealing and extension (or amplification) step temperature of the PCR. If the extension (or amplification) step temperature is lower than 55 ° C, the specificity of the PCR product may decrease. If the annealing and extension (or amplification) step temperature is higher than 74 ° C, extension by the primer may not occur. The temperature of the annealing and extension (or amplification) step may be 55 ° C to 75 ° C, preferably 72 ° C.

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

The PCR device according to the third embodiment of the present invention is capable of moving left and right and / or up and down by the driving means 500a on the first column block 100a and the second column block 200a, And a chip holder 300a mounted thereon. The chip holder 300a is a module in which the PCR chip 10 is mounted in the PCR device. The inner wall of the chip holder 300a is fixedly mounted on the outer wall of the PCR chip 10 so that the PCR chip 10 does not separate from the chip holder 300a when the nucleic acid amplification reaction is performed by the PCR device Shape and structure. The chip holder 300a is operatively connected to the driving means 500a. In addition, the PCR chip 10 may be removably attached to the chip holder 300a.

The driving means 500a includes all the means for making the chip holder 300a equipped with the PCR chip 10 movable left and right and / or up and down on the first column block 100a and the second column block 200a . The chip holder 300a on which the PCR chip 10 is mounted can reciprocate between the first column block 100a and the second column block 200a by moving the driving means 500a in the left- The chip holder 300a on which the PCR chip 10 is mounted can be brought into contact with and separated from the first column block 100a and the second column block 200a by the vertical movement of the driving means 500a have. The driving means 500a of the PCR apparatus according to the third embodiment of the present invention shown in Fig. 3a includes a rail 510a extending in the left-right direction and a rail 510a slidably movable in the left-right direction through the rail 510a And a connecting member 520a slidable in the vertical direction, and the chip holder is disposed at one end of the connecting member 520a. The right and / or left and / or up and down movement of the driving means 500a can be controlled by control means (not shown) arranged to be drivable inside or outside the PCR apparatus, It is possible to control the contact and separation between the chip holder 300a on which the PCR chip 10 is mounted and the first column block 100a and the second column block 200a for the annealing and extension (or amplification) have.

FIG. 3B shows each step of the nucleic acid amplification reaction by moving the chip holder of the PCR apparatus according to the third embodiment of the present invention. The nucleic acid amplification reaction by the PCR apparatus is performed by the following steps. First, an oligonucleotide primer, a DNA polymerase, a deoxyribonucleotide triphosphate (dNTP), an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, a DNA polymerase, , A PCR buffer (PCR buffer), and mounting the PCR chip 10 to the chip holder 300a. Thereafter, or concurrently, the first thermal block 100a is heated and maintained at a temperature for the denaturation step, for example 90 ° C to 100 ° C, and preferably heated and maintained at 95 ° C. The second thermal block 200 is heated and maintained at a temperature for annealing and extending (or amplifying), for example, 55 ° C to 75 ° C, and preferably heated and maintained at 72 ° C . The PCR chip 10 is moved downward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a on which the PCR chip 10 is mounted is inserted into the first column block 100a) to perform a first denaturation step of the PCR (step x). Thereafter, the PCR chip 10 is moved upward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a, on which the PCR chip 10 is mounted, 100a to terminate the first denaturation step of the PCR and control the connecting member 520a of the driving means 500a to move the PCR chip 10 above the second column block 200a (Step y). Then the PCR chip 10 is moved downward by controlling the connecting member 520a of the driving means 500a so that the chip holder 300a on which the PCR chip 10 is mounted is inserted into the second column block 100a) to perform a first annealing and extension (or amplification) step of the PCR (step z). Finally, the connection member 520a of the driving means 500a is controlled to move the PCR chip 10 upward, and the chip holder 300a, on which the PCR chip 10 is mounted, The PCR chip 10 is separated from the first column block 100a by controlling the connecting member 520a of the driving means 500a by terminating the first annealing and extending And repeating the above steps x, y, and z to perform a nucleic acid amplification reaction (circulation step).

FIG. 3C shows a step of observing nucleic acid amplification reaction in real time using the PCR apparatus according to the third embodiment of the present invention. The PCR apparatus according to the third embodiment of the present invention further includes a light source 700a disposed between the first column block 100a and the second column block 200a and the light source 700a A light detecting portion 800a for detecting light emitted from the light source 700a is further disposed or a light detecting portion 800b for detecting light emitted from the light source 700a between the first column block 100a and the second column block 200a A light detecting unit 800a may be further disposed and a light source 700a may be further disposed on the chip holder 300a. The light detecting unit 800a is disposed on the driving unit 500a and the driving unit 900a may include a penetrating unit 530a for passing light emitted from the light source 700a. In addition, the PCR chip 10 may be a light-transmitting material, specifically, a light-transmitting plastic material.

The degree of amplification of the nucleic acid in the PCR chip 10 during the nucleic acid amplification reaction by the PCR device 1 can be detected in real time by the arrangement of the light source 700a and the photodetector 800a. In order to detect the amplification degree of the nucleic acid in the PCR chip 10, a separate fluorescent material may be further added to the sample solution to be introduced into the PCR chip 10. The light source 700a is disposed as widely as possible in a spaced space between the first column block 100a and the second column block 200a and is arranged to emit as much light as possible. The light source 700a may be drivingly connected to a lens (not shown) that collects light emitted from the light source 700a and an optical filter (not shown) that filters light of a specific wavelength band.

The step of detecting the amplification degree of the nucleic acid in the PCR chip 10 in real time during the nucleic acid amplification reaction by the PCR apparatus according to the third embodiment of the present invention is performed by the following steps. After the completion of the first denaturation step of the PCR, the connection member 520a of the driving unit 500a is controlled to move the PCR chip 10 from the top of the first column block 100a to the top of the second column block 200a Or controlling the connection member 520a of the driving means 500a after the first annealing and extension (or amplification) step of the PCR is completed to transfer the PCR chip 10 to the second column block 200a The chip holder 300a on which the PCR chip 10 is mounted is controlled by the connecting member 520a of the driving means 500a to move the chip holder 300a on the first column block 200a, And then stopping on a spaced space between the column block 100a and the second column block 200a. Thereafter, light is emitted from the light source 700a, and the emitted light passes through the light-transmissive PCR chip 10, specifically, the reaction channel of the PCR chip 10, in this case, the reaction channel) The optical detecting unit 800a detects an optical signal generated by the amplification of the nucleic acid in the sample. In this case, light passing through the light-transmissive PCR chip 10 may pass through the driving unit 500a, specifically, the penetration unit 530a disposed in the rail 510a and reach the light output unit 800a have. Therefore, according to the PCR apparatus according to the third embodiment of the present invention, the reaction result by amplification of the nucleic acid (the fluorescent substance is bound) in the reaction channel during each circulation step of the PCR is monitored in real time The amount of the target nucleic acid contained in the initial reaction sample can be measured and analyzed in real time.

4A to 4I are views showing a PCR apparatus according to a fourth embodiment of the present invention.

4A shows a light-transmissive column block 100b included in the PCR apparatus according to the fourth embodiment of the present invention. The PCR apparatus includes a substrate 10b, a heat generating layer 20b including conductive nanoparticles disposed on the substrate 10b, an insulating protective layer 30b disposed on the heat generating layer, A light-transmissive thermal block 100b having an electrode 40b formed thereon and having optical transparency; And a PCR chip 10 according to a second embodiment of the present invention arranged so as to be able to contact the upper surface of the light-transmitting heat block 100b.

The substrate 10b may be a light-transmissive plate material, and may be a light-transmitting glass or a light-transmitting plastic material. The heating layer 20b serves as a heat source for the optically transparent thermal block 100b for performing the denaturation step of the PCR, the annealing step, and the extension (or amplification) step. 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. Since the conductive nanoparticles are dispersed in the solvent, the conductive nanoparticles can be easily laminated on the substrate 10b. Therefore, the thickness of the heating layer 20b can be easily controlled by adjusting the number of the layers . Also, the conductivity of the heating layer 20b can be easily controlled by adjusting the concentration of the dispersion liquid containing the conductive nanoparticles. An adhesion strengthening layer (not shown) may be formed between the substrate 10b and the heating layer 20b to strongly fix the heating layer 20b to the substrate 10b. The adhesion-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 20b 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 protective layer 30b is for physically and / or electrically protecting the heat generating layer 20b, 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 30b may be transparent. The electrode 40b is directly or indirectly connected to the heating layer 20 to supply power to the heating layer 20b. The electrode 40b 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. 4A, the electrodes 40b are connected to both sides of the heating layer 20b. However, if the heating layer 20b can supply power to the heating layer 20b, the electrodes 40b can be connected and arranged in various operable positions. In addition, the electrode 40b may be electrically connected to a power source included in the PCR apparatus or disposed externally. For example, the electrode 40b directly contacts the heating layer 20b and connects the heating layer 20b to an external circuit (not shown) via a wiring (not shown) The terminals can be arranged to be stably fixed to the electrode 40b.

The light-transmissive thermal block 100b includes a chip contact portion 50b with which a PCR chip (not shown) contacts at least a part of the upper surface thereof. The PCR chip 10 may be heated or cooled according to the heat supply or recovery of the light-transmitting thermal block 100b by contacting the chip contact portion 50b to perform each step of the PCR. In addition, the PCR chip 10 may directly or indirectly contact the chip contact portion 50. The PCR device including the light-transmissive thermal block 100b has many advantages over the conventional PCR device using a conventional calc-heater, a ceramic heater, or a metal heater as a heat block. 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 when the substrate 10b, the heat generating layer 20b, and the insulating protective layer 30b are fabricated, they can be included in the sample solution when implemented together with the optical providing and photo- Real-time monitoring of the PCR by the fluorescent material is possible. Since the thickness of the substrate 10b, the heat-generating layer 20b and the insulating protective layer 30b can be easily controlled, the light-transmitting thermal block 100b can be slim, It is possible to downsize the PCR apparatus including the block 100b. In addition, since the conductive nanoparticles are uniformly distributed in the heating layer 20b to uniformly distribute the heat of the light-transmitting thermal block 100b and to control the temperature quickly, the detection efficiency of the PCR result is high, You can get it quickly. The uniformity of the thermal distribution of the light-transmissive column block 100b and the rapidity of the temperature control can be confirmed as an experimental result according to FIG. FIG. 4B shows a temperature change with time of the light-transmitting thermal block 100b included in the PCR apparatus according to the fourth embodiment of the present invention. In the conventional PCR apparatus, electric power is applied to a calcite heater, a ceramic heater or a metal heater used as a heat block to observe a temperature distribution, and electric power is applied to the light-transmitting column block 100b through the electrode 40b, Respectively. As a result, although the temperature distribution on the conventional heater is not uniform over the heater surface, the temperature distribution on the light-transmitting heat block 100b is observed to be uniform as a whole compared to the conventional heater. In addition, power was applied to the light-transmissive column block 100b through the electrode 40b to observe the temperature change of the light-permeable column block 100b with 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) .

4C shows a light-transmitting thermal block 100b included in the PCR device according to the fourth embodiment of the present invention in which the light absorbing layer 60b is disposed in contact with the lower surface of the substrate 10b, Transmitting thermal block 100b included in the PCR apparatus according to the fourth embodiment of the present invention in which the light reflection preventing layer 70b is disposed in contact with the upper surface of the substrate 10b, And a light reflection preventing layer 70b for preventing light reflection due to contact between the external air layer and the insulating protecting layer 30b is disposed in contact with the upper portion of the insulating protecting layer 30b And shows a light-transmissive column block 100b included in the PCR apparatus according to the fourth 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 100b has light transmittance as a whole, most of the excitation light originating from the light source is transmitted as it is, thereby enhancing the sensing efficiency of the optical signal. However, a part of the excitation light may be reflected on the light-transmissive column block 100b or may be reflected after passing through the light-transmissive column block 100b to act as noise of the optical signal. Accordingly, the light absorbing material may be treated on the lower surface of the light-transmitting heat block 100b to further enhance the sensing efficiency. According to Fig. 4C, a light absorbing layer 60b is disposed in contact with the lower surface of the substrate 10b, and the light absorbing layer 60b includes a light absorbing material. The light absorbing substance may be, for example, mica, but is not limited as long as it is a substance having a property of absorbing light. Therefore, a part of the light derived from the light source is absorbed by the light absorbing layer 60b, and generation of reflected light acting as noise of the optical signal can be suppressed to the utmost. Alternatively, the light reflection preventing material may be treated on the upper surface of the light-transmitting heat block 100b to further enhance the sensing efficiency. 4D, a light reflection preventing layer 70b is disposed in contact with the upper surface of the insulating protection layer 30b, and the light reflection preventing layer 70b is combined with the insulating protection layer 30b, 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. More preferably, the light absorbing material is treated on the lower surface of the light-transmitting heat block 100b and the light reflection preventing material is treated on the upper surface of the light-transmitting heat block 100b 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 typical metallic material is about 20 to 80%, but the light-transmitting heat block (not shown) including the light absorbing layer 60b or the antireflection layer 70b according to FIG. 4C or FIG. The light reflectance of the light-transmitting heat block 100b according to the present invention including the light absorbing layer 60b and the light reflection preventing layer 70b according to FIG. 4e can be reduced to 0.2% to 4% The light reflectance can be reduced to 0.2% or less.

4f shows that the PCR chip 10 is disposed on the light-transmitting column block 100b of the PCR apparatus according to the fourth embodiment of the present invention including the optical coupler and the optical detector. According to FIG. 4F, the PCR apparatus further includes a light supplier 200b disposed to be operable to provide light to the PCR chip 10 disposed in the chip contact portion 50b, and a PCR provided in the chip contact portion 50b. And a light detecting portion 300b disposed to be capable of receiving light emitted from the chip 10. [ The optical detector 200b is a module for providing light to the PCR chip 10. The optical detector 300b receives light emitted from the PCR chip 10 and outputs the light to the PCR chip 10 This is a module for measuring the PCR reaction to be performed. Light is emitted from the light supplier 200b and the emitted light passes through or reflects the reaction channel (not shown) of the PCR chip 10, specifically, the PCR chip 10, The optical detecting unit 300b detects an optical signal generated by nucleic acid amplification in the reaction channel. Therefore, according to the PCR apparatus of the fourth embodiment of the present invention, the amplification of the nucleic acid (in which the fluorescent substance is bound) in the reaction channel during each circulation step of the PCR in the PCR chip 10 By monitoring the reaction results in real time, it is possible to measure and analyze in real time the amplification and amplification degree of the target nucleic acid contained in the initial sample solution. In addition, the light providing part 200b and the light detecting part 300b may be disposed on the top or bottom of the light-transmitting column block 100b, or may be disposed on the light-blocking block 100b. However, the arrangement of the optical coupler 200b and the optical detector 300b may be varied in consideration of the arrangement relationship with other modules for optimal implementation. Preferably, 200b and the photodetector 300b may be disposed above the light-transmitting column block.

Referring to FIG. 4D, the light providing unit 200b includes a light emitting diode (LED) light source or a laser light source 210b, a first light filter (not shown) for selecting light having a predetermined wavelength from the light emitted from the light source And a first optical lens 240b for collecting the light emitted from the first optical filter 230b and a second optical lens 240b for emitting light between the light source 210b and the first optical filter 230b. 1 aspheric lens 220b. The light source 210b includes all light sources capable of emitting light, and includes a light emitting diode (LED) light source or a laser light source. The first optical filter 230b 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 210b. For example, the first optical filter 230b can pass only light having a wavelength of 500 nm or less among the light emitted from the light source 210b. The first optical lens 240b collects the incident light and increases the intensity of the emitted light. The first optical lens 240b increases the intensity of the light irradiated to the PCR chip 10 through the light-transmitting column block 100b . The optical fiber 200b further includes a first aspherical lens 220b arranged to spread light between the light source 210b and the first optical filter 230b. By adjusting the arrangement direction of the first aspherical lens 220b, the light range emitted from the light source 210b is expanded to reach the measurable area.

4H, the photodetector 300b includes a second optical lens 310b for collecting light emitted from the PCR chip 10 disposed on the chip contact portion 50b, a second optical lens 310b for emitting light emitted from the second optical lens, A second optical filter 320b for selecting light having a predetermined wavelength in the first optical fiber 320b and an optical analyzer 350b for detecting an optical signal from light emitted from the second optical fiber 320b, The second aspherical lens 330b disposed between the filter 320b and the optical analyzer 350b to collect light emitted from the second optical filter 320b and the second aspherical lens 330b, (330b) disposed between the first aspheric lens (330b) and the optical analyzer (350b) to remove noise of light emitted from the second aspherical lens (330b) and amplify light emitted from the second aspherical lens a photodiode integrated circuit (PDIC) 340b, The. The second optical lens 310b collects the incident light and increases the intensity of the emitted light. The intensity of light emitted from the PCR chip 10 is increased through the light-transmitting column block 100b Thereby facilitating optical signal detection. The second optical filter 320b selectively emits light of a specific wavelength among incident light having various wavelength ranges and is configured to emit light according to the wavelength of the predetermined light emitted from the PCR chip 10 through the light- And can be variously selected. For example, the second optical filter 320b may pass only the light having a wavelength of 500 nm or less of the predetermined light emitted from the PCR chip 10 through the light-transmitting column block 100b. The optical analyzer 350b is a module for detecting an optical signal from the light emitted from the second optical filter 320b and is capable of performing qualitative and quantitative measurement by converting light emitted from the sample solution into electrical signals . The optical detector 300b includes a second aspherical lens 330b disposed between the second optical filter 320b and the optical analyzer 350b to collect light emitted from the second optical filter 320b, As shown in FIG. By adjusting the arrangement direction of the second aspherical lens 330b, the light range emitted from the second optical filter 320b is expanded to reach the measurable area. The photodetector 300b may remove noise of light emitted from the second aspherical lens 330b between the second aspherical lens 330b and the optical analyzer 350b, And a photodiode integrated circuit (PDIC) 340b arranged to amplify light emitted from the lens 330b. By using the photodiode integrated device 340b, it is possible to further miniaturize the PCR device, minimize the noise, and measure the reliable optical signal.

According to FIG. 4i, the PCR device adjusts the traveling direction of light so that the light emitted from the light providing part 200b reaches the optical detecting part 300b, and one or more And further includes dichroism filters 400x and 400y. The dichroic filters 400x and 400y are modules that selectively transmit or selectively reflect light according to wavelengths. According to FIG. 4K, the dichroic filter 400x is arranged at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light supplier 200b, and selectively transmits the short wavelength component according to the wavelength, Is reflected at a right angle to reach the PCR chip 10 disposed on the light-transmitting column block 100b. The dichroic filter 400y is disposed at an angle of about 45 degrees with respect to the optical axis of the light reflected from the PCR chip 10 and the light-transmitting column block 100b. The light is selectively incident on the short wavelength component And causes the long wavelength component to be reflected at a right angle to reach the optical detector 300b. The light reaching the photodetector 300b is converted into an electric signal by the optical analyzer to indicate the amplification and amplification of the nucleic acid.

5A to 5I are views showing a PCR apparatus according to a fifth embodiment of the present invention.

5A to 5I, a PCR apparatus according to a fifth embodiment of the present invention includes a heater group having at least one heater, at least two heater groups, and at least two heater groups are spaced apart from each other A thermal block having a contact surface of the PCR chip on which at least one surface of the target unit is accommodated; An electrode unit having an electrode connected to supply electric power to the heaters provided in the column block; And a PCR chip 10 according to the second embodiment of the present invention, which is disposed so as to be able to make contact with the thermal block so that heat exchange with one or more heaters included in the thermal block is possible.

The thermal block 100c is a module implemented to supply heat to a target sample at a specific temperature to perform PCR. The thermal block 100c includes a contact surface of the PCR chip 10 on which at least one surface of the target sample is received, The PCR is performed by supplying heat to a target sample existing in at least one reaction channel in contact with one surface of the chip 10. The thermal block 100c is based on a substrate. The substrate is implemented with any material that does not change its physical and / or chemical properties due to heating and temperature maintenance of the heater disposed within the substrate and does not cause mutual heat exchange between two or more heaters spaced apart within the substrate . For example, when the PCR apparatus according to the fifth embodiment of the present invention is used for real-time PCR, the substrate may be made of plastic, glass, silicon, or the like, And is preferably made of a transmissive material. The thermal block 100c may have a generally planar shape, but is not limited thereto. The heat block 100c includes two or more heater groups including one or more heaters and two or more heater groups. The two or more heater groups are repeatedly disposed two or more apart from each other so as to avoid mutual heat exchange. The contact surface of the PCR chip 10 may be implemented on at least one surface of the thermal block 100c and may be implemented in various shapes for efficiently supplying heat to the PCR chip 10 containing the target sample , And a planar shape or a pillar shape is preferable such that the surface area of the contact surface is wide. The heaters 111c, 112c, 121c, 122c, 131c, and 132c are heat generating elements, and heat wires (not shown) may be disposed therein. The hot wire may be drivably connected to various heat sources to maintain a constant temperature, and may be drivably connected to various temperature sensors for monitoring the temperature of the hot wire. The heating line may be arranged to be symmetrical in the up and down and / or the left and right directions with respect to the surface center point of the heater in order to maintain the internal temperature of the heater as a whole. Also, a thin film heater (not shown) may be disposed inside the heater. The thin film heater may be disposed at regular intervals in the up and down and / or the left and right directions with respect to the center point of the surface of the heater in order to maintain the internal temperature of the heater as a whole. The heater may also be a heating element, a metal material itself, such as chromium, aluminum, copper, iron, silver, etc., for even heat distribution and rapid heat transfer to the same area. The heater may be a light-transmitting heat generating element, for example, an oxide semiconductor material or a conductive nanoparticle including a substance to which an impurity selected from the group consisting of In, Sb, Al, Ga, C and Sn is added to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive high molecular materials, carbon nanotubes, and graphene. If the PCR device is used for real-time PCR, the heater is preferably a light-transmitting heating element. The heater groups 110c, 120c, and 130c are units that include the one or more heaters, and maintain the temperature for performing the denaturation step, the annealing step, and / or the extension step for performing the PCR. More than two heaters are disposed in the heat block 100c, and the two or more heaters are spaced apart from each other such that mutual heat exchange does not occur. The heater group may include two to four heaters in the heat block 100c. That is, the heat block includes two heater groups, the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing / extension step temperature, or the first heater group The PCR annealing / extension step temperature can be maintained and the second heater group can maintain the PCR denaturation step temperature. The heat block includes three heater groups, the first heater group maintains the PCR denaturation temperature, the second heater group maintains the PCR annealing temperature, and the third heater group maintains the PCR extension temperature , Or the first heater group maintains the PCR annealing step temperature, the second heater group maintains the PCR extension step temperature, the third heater group maintains the PCR denaturation step temperature, or the first heater group Group can maintain the PCR extension step temperature, the second heater group can maintain the PCR denaturation step temperature, and the third heater group can maintain the PCR annealing step temperature. Preferably, the heater group is disposed three times in the thermal block 100c to maintain the temperature for performing PCR in three stages, i.e., the denaturation step, the annealing step and the extension step, The heater group may be disposed twice in the thermal block 100c to maintain the temperature for performing PCR in two stages, i.e., a denaturation step and an annealing / extension step, but is not limited thereto. When the heater group is disposed in the thermal block 100c twice and performs two steps for performing the PCR, that is, the denaturation step and the annealing / extending step, there are three steps for PCR execution: denaturation step, annealing step and extension step It is possible to shorten the reaction time and simplify the structure by reducing the number of heaters. In this case, in the third step for carrying out PCR, the temperature for carrying out the denaturation step is 85 ° C to 105 ° C, preferably 95 ° C, and the temperature for carrying out the annealing step is 40 ° C to 60 ° C, preferably 50 ° C., and the temperature for carrying out the extension step is 50 ° C. to 80 ° C., preferably 72 ° C. In the second step for carrying out the PCR, the temperature for carrying out the denaturation step is 85 ° C. to 105 ° C., 95 < 0 > C, and the temperature for carrying out the annealing / lengthening step is from 50 [deg.] C to 80 [deg.] C, preferably 72 [deg.] C. However, the specified temperature and temperature range for performing the PCR can be adjusted within a range that can be realized in performing PCR. The heater group may further include a heater for performing a temperature buffering function. The heater unit (10c, 20c) is a unit including the at least two heater groups including the at least one heater, and includes a denaturation step for performing PCR, to be. The heater unit is repeatedly disposed in the heat block 100c at least two times. Preferably, the heater unit may be repeatedly disposed in the heat block 100c 10 times, 20 times, 30 times, or 40 times. However, the present invention is not limited thereto.

5A, the heating block 100c includes heater units 10c and 20c repeatedly arranged, two heater groups 110c and 120c respectively included therein, and one heater 111c and 121c respectively included therein. Thereby sequentially providing a two-step temperature for performing the PCR, that is, one temperature of the denaturation step and one temperature of the annealing / extending step. For example, the first heater 111c is maintained at a temperature of 85 deg. C to 105 deg. C, preferably at 95 deg. C, so that the first heater group 110c provides a temperature for performing the denaturation step, 2 heater 121c is maintained at 1 temperature, preferably 72 ° C, in the range of 50 ° C to 80 ° C so that the second heater group 120c provides the temperature for performing the annealing / 100c sequentially provide a second-stage temperature for PCR execution in the first heater unit 10c and the second heater unit 20c.

5B, the thermal block 100c includes heater units 10c and 20c repeatedly arranged, two heater groups 110c and 120c respectively included therein, and two heaters 111c, 112c, 121c, and 122c to sequentially provide two temperatures for performing PCR, that is, two temperatures of the denaturation step and two annealing / extending steps. For example, the temperature of the first heater 111c is 1 temperature in the range of 85 deg. C to 105 deg. C, and the temperature of the second heater 112c is in the range of 85 deg. C to 105 deg. , The first heater group 110c provides a temperature for performing the denaturation step, the third heater 121c provides one temperature in the range of 50 ° C to 80 ° C, the fourth heater 122c provides a temperature for performing the denaturation step, The second heater group 120c maintains a temperature equal to or different from the temperature of the third heater 121c in the range of 80 占 폚 to provide the temperature for performing the annealing / The second heater unit 20c sequentially provides a second temperature for PCR execution in the first heater unit 10c and the second heater unit 20c.

5c, the thermal block 100c includes heater units 10c and 20c repeatedly arranged, three heater groups 110c, 120c and 130c respectively included therein, and one heater 111c, 121c, and 131c to sequentially provide a three-step temperature for PCR execution, that is, one temperature in the denaturation step, one temperature in the annealing step, and one temperature in the extension step. For example, the first heater 111c is maintained at a temperature of 85 deg. C to 105 deg. C, preferably at 95 deg. C, so that the first heater group 110c provides a temperature for performing the denaturation step, 2 heater 121c is maintained at a temperature of 40 ° C to 60 ° C, preferably at 50 ° C, so that the second heater group 120c provides a temperature for performing the annealing step, and the third heater 131c, The first heater group 130c maintains the temperature of 1 to 50 ° C to 80 ° C, preferably 72 ° C, so that the third heater group 130c provides the temperature for performing the extension step, And the three-step temperature for performing the PCR in the first heater unit 10c and the second heater unit 20c.

5d, repeatedly arranged heater units 10c and 20c, three heater groups 110c, 120c and 130c respectively included therein and two heaters 111c, 112c, 121c, 122c and 131c , 132c to sequentially provide the three-step temperature for performing the PCR, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step. For example, the temperature of the first heater 111c is set to 1 at a temperature of 85 deg. C to 105 deg. C, and the temperature of the second heater 112c is set to be equal to or different from the temperature of the first heater 111c The first heater group 110c provides a temperature for performing the denaturation step, the third heater 121c has a temperature of 1 to 40 ° C to 60 ° C, the fourth heater 122c has a temperature of 40 ° C to 120 ° C, The second heater group 120c provides a temperature for performing the annealing step by maintaining one temperature that is the same as or different from the temperature of the third heater 121c in the range of 60 占 폚 and the fifth heater 131c provides 50 占 폚 And the third heater group 130c is maintained at a temperature that is the same as or different from the temperature of the fifth heater 131c in the range of 50 占 폚 to 80 占 폚, The thermal block 100c performs PCR in the first heater unit 10c and the second heater unit 20c A third step for temperature provides successively repeated.

As shown in FIGS. 5A to 5D, by repeatedly arranging two or more heaters that maintain a constant temperature, the rate of temperature change can be greatly improved. For example, according to the conventional single heater method adopting only one heater, the rate of temperature change is within a range of 3 ° C. to 7 ° C. per second, whereas according to the repetition heater arrangement method according to the fifth embodiment of the present invention, The rate of temperature change between the heaters can be in the range of 20 ° C to 40 ° C per second, greatly reducing the reaction time. In the nucleic acid amplification reaction in which the heaters are spaced apart from each other so that mutual heat exchange does not occur and as a result, they can be greatly affected even by a minute temperature change, the denaturation step, the annealing step and the extension step (or the denaturation step and annealing / Denaturation step), and it is possible to maintain a desired temperature or a temperature range only at a position where heat is supplied from the heaters. In the thermal block 100c, two or more heater units are repeatedly arranged, and the number of repeated arrangements of the heater units 10c and 20c may vary depending on the type of the user or target sample to be subjected to the PCR . For example, in a case where the PCR apparatus according to the fifth embodiment of the present invention is applied to PCR in which the circulation cycle is 10 cycles, the heater unit can be repeatedly arranged 10 times. That is, the heater unit can be repeatedly arranged 10 times, 20 times, 30 times, 40 times, 50 times, etc. in consideration of the PCR cycle period depending on the type of the user or the target sample to be subjected to PCR, It is not. On the other hand, the heater unit may be repeatedly arranged with a half number of a predetermined PCR cycle period. For example, when the PCR apparatus according to an embodiment of the present invention is applied to PCR in which the circulation cycle is 20 cycles, the heater unit can be repeatedly arranged 10 times. In this case, the target sample solution is repeatedly subjected to PCR cycles 10 times in the direction from the inflow part 304c to the outflow part 305c in the reaction channel 303c disposed in the nucleic acid amplification part 300c to be described in detail below , And thereafter, the PCR circulation cycle can be repeated 10 times in the direction from the outflow portion 305c to the inflow portion 304c.

FIG. 5E shows the structure of a PCR apparatus according to the fifth embodiment of the present invention including the PCR chip 10. FIG. 5E shows a lateral cross-sectional view of the PCR chip 10 and the thermal block 100c of the PCR device according to the fifth embodiment of the present invention, and the lower part of FIG. 5E shows the fifth embodiment of the present invention An upper plan view of the PCR chip 10 according to the example and the thermal block 100c of the PCR device.

Referring to FIG. 5E, the heat block 100c includes a heater unit disposed repeatedly 10 times, and the heater unit includes a first heater group and a second heater group, and the first heater group and the second heater group One heater, i.e., a first heater 110c and a second heater 120c, respectively. The heater, the heater group, the heater unit and the heat block according to Fig. 5E are as described above.

The electrode unit 200c is a module that receives power from a power supply unit (not shown) and supplies power to the heat block 100c so as to generate heat, and supplies power to the heaters provided in the heat block 100c And electrodes 210c and 220c connected to each other. The first electrode 210c of the thermal block 100c is connected to supply power to the first heater 110c and the second electrode 220c is connected to the second heater 120c, However, the present invention is not limited thereto. The electrode unit 200c may be disposed outside the thermal block 100c. If the temperature of the first heater 110c is maintained at a PCR denaturation temperature, for example, 85 ° C to 105 ° C, and the temperature of the second heater 120c is maintained at a PCR annealing / The first electrode 210c is supplied with power for maintaining the PCR denaturation step temperature from the power supply unit and the second electrode 220c is supplied with power for maintaining the PCR annealing / have. The first electrode 210c and the second electrode 220c may be connected to the first heater 110c and the second heater 120c repeatedly disposed in the thermal block 100c. The first electrode 210c and the second electrode 220c may be made of a conductive material such as gold, silver, or copper, and are not particularly limited.

The PCR chip 10 is brought into contact with the thermal block 100c so that heat can be exchanged with the thermal block 100c and heat is supplied from the first heater 110c and the second heater 120c have. The PCR chip 10 may further include one or more reactions extending in the longitudinal direction to correspond to the upper portion 301c of the first heater 110c and the upper portion 302c of the second heater 120c, Channel 14 may be provided. 5E, the reaction channel 14 is formed in a position corresponding to the upper portion corresponding portion 301c of the first heater 110c disposed in the thermal block 100c and the upper corresponding portion 301c of the second heater 120c 302c are fluidly connected and extend in a longitudinal direction. The upper side corresponding portion 301c of the first heater 110c of the reaction channel 14 is a region where a PCR denaturing reaction of the nucleic acid existing in the target sample takes place and the upper side corresponding portion 302c of the second heater 120c ) May be the region where the PCR annealing / extension reaction of the nucleic acid present in the target sample takes place. That is, PCR is performed while the target sample flowing through the reaction channel 14 passes through the upper side corresponding portion 301c of the first heater 110c and the upper side corresponding portion 302c of the second heater 120c successively . The PCR chip 10 may include an inlet 15 and an outlet 16 at both ends of the at least one reaction channel 14. 5E, the one or more reaction channels 14 are disposed on the upper side corresponding portion 301c of the first heater 110c disposed at the best position among the heater units and the upper side corresponding portion 301c of the second heater 120c disposed last It is preferable to extend and extend so as to pass the corresponding portion 302c in the straight length direction. Therefore, the target sample flowing through the inlet 15 passes through the reaction channel 14 in the longitudinal direction while the upper portion 301c of the first heater 110c and the upper portion 301c of the second heater 120c And the PCR denaturation step and the PCR annealing / extension step are repeatedly performed in the corresponding part 302c, respectively, and may be discharged to the outside through the outflow part. On the other hand, the PCR chip 10 may have a planar shape as a whole, but is not limited thereto. If the PCR device according to the fifth embodiment of the present invention is used for real-time PCR, the PCR chip 10 may be implemented as a light- And is preferably made of a transmissive material.

5f shows a PCR apparatus according to the fifth embodiment of the present invention including a PCR chip, a power supply, and a pump.

Referring to FIG. 5F, the PCR apparatus according to the fifth embodiment of the present invention includes a PCR chip 10, a power supply unit 400c, and a pump 500c. Specifically, the PCR chip 10 is placed in contact with the column block. The PCR chip 10 and its constituent elements are the same as those already described. The power supply unit 400c is a module for supplying power to the electrode unit 200c and may be connected to the first electrode 210c and the second electrode 220c of the electrode unit 200c. For example, when the PCR chip 10 is placed in contact with the thermal block for PCR, a first power port (not shown) of the power supply 400c is electrically connected to the first electrode 210c And a second power port (not shown) of the power supply unit 400c is electrically connected to the second electrode 220c. When there is a user instruction to perform PCR, the power supply unit 400c supplies power to the first electrode 210c and the second electrode 220c, respectively, and supplies power to the first heater 110c and the second heater 110c of the column block, The second heater 120c can be rapidly heated and when the respective heaters 110c and 120c reach a predetermined temperature, the power supply amount is controlled to maintain the predetermined temperature. For example, the predetermined temperature may be a PCR denaturation temperature (85 ° C to 105 ° C, preferably 95 ° C) in the first heater 110c and a PCR annealing / extension temperature (in the second heater 120c) 50 ° C to 80 ° C, preferably 72 ° C) or the PCR annealing / extension step temperature (50 ° C to 80 ° C, preferably 72 ° C) and the second heater 120c in the first heater 110c, The PCR denaturation step temperature (85 ° C to 105 ° C, preferably 95 ° C).

The pump 500c is a module for controlling the flow rate and the flow rate of the fluid flowing in the at least one reaction channel 14 of the PCR chip 10 and may be a positive pressure pump or a negative pressure pump, a syringe pump. The pump 500c may be drivably disposed on a portion of the reaction channel 14 but preferably includes an inlet 15 and / or an outlet 16 formed at both ends of the reaction channel 303c. ). The pump 500c not only serves as a pump when the pump 500c is connected to the inlet 15 and / or the outlet 16 but also serves as a pump through the inlet 15 and / It may also act as a stopper to prevent solution from escaping. In order to control the flow rate and the flow rate of the fluid flowing in the reaction channel 14, that is, the target sample solution in one direction, the pump 500c is connected to the inflow section 15 and the outflow section 16 In order to control both the flow rate and the flow rate of the fluid flowing in the reaction channel 14, that is, the target sample solution in both directions, the pump (not shown) 500c may be connected to both the inlet (15) and the outlet (16).

The nucleic acid amplification reaction of the target sample in the PCR apparatus including the PCR chip 10, the electric power supply unit 400c and the pump 500c may be performed through the following steps as an embodiment.

1. The desired double-stranded target DNA, an oligonucleotide primer having a sequence complementary to the specific nucleotide sequence to be amplified, DNA polymerase, deoxyribonucleotide triphosphates (dNTP), PCR reaction buffer Prepare the target sample solution containing.

2. The target sample solution is introduced into the PCR chip 10. In this case, the target sample solution is placed in the reaction channel 13 inside the PCR chip 10 through the inlet 15.

3. The electrode unit 200c, specifically, the first electrode 210c and the second electrode 220c are connected to the power supply unit 400c and the inlet unit 14 of the PCR chip 10, And the outlet (15) with the pump (500c).

4. The power supply unit 400c is instructed to supply electric power to heat the first heater 110c and the second heater 120c through the first electrode 210c and the second electrode 220c, The PCR denaturation step temperature (95 ° C) in the case of the first heater 110c and the PCR annealing / elongating step temperature (72 ° C) in the case of the second heater 120c are maintained.

5. If positive pressure is provided by the pump 500c connected to the inlet 15 or negative pressure is provided by the pump 500c connected to the outlet 16, So as to flow in the horizontal direction from the inside. In this case, the flow rate and the flow rate of the target sample solution can be controlled by adjusting the intensity of the positive pressure or the negative pressure provided by the pump 500c.

By performing the above steps, the target sample solution is supplied to the upper portion 301c of the first heater 110c from the end of the inlet portion 15 of the reaction channel 14 to the end of the outlet portion 16, PCR is performed while moving the upper corresponding portion 302c of the heater 120c in the longitudinal direction. Referring to FIG. 5E, the target sample solution is supplied with heat from the heat block 100c in which the heater unit including the first heater 110c and the second heater 120c are repeatedly arranged ten times, The PCR cycling cycle is completed through the PCR denaturation step at the upper corresponding part 301c of the first heater 120c and the PCR annealing / extension step at the upper corresponding part 302c of the second heater 120c. Optionally, the target sample solution further comprises an upper corresponding portion 301c of the first heater 110c from the end of the outlet 16 of the reaction channel 14 to an end of the inlet 15, It is possible to re-execute the PCR while reversing the upper-side corresponding portion 302c of the upper portion 120c in the longitudinal direction.

FIG. 5G shows a PCR device according to a fifth embodiment of the present invention including a PCR chip of a light-transmitting material and a thermal block, a power supply, a pump, and a light detecting part including the light source disposed between the first heater and the second heater, / RTI >

5g, the PCR apparatus according to the fifth embodiment of the present invention includes a light-transmitting material, a PCR chip 10, and a light source 150c between the first heater 110c and the second heater 120c A heat block 100c arranged therein, a power supply 400c, a pump 500c, and a photodetector 600c. The power supply unit and the pump have already been described.

The PCR chip 10 according to FIG. 5G is formed of a light transmitting material and a light source 150c is disposed between the first heater 110c and the second heater 120c of the thermal block 100c . In addition, the PCR apparatus further includes a photodetector 600c for detecting light emitted from the light source 150c. Accordingly, the PCR apparatus according to the fifth embodiment of the present invention according to FIG. 5g can measure and analyze the nucleic acid amplification process in real time when PCR is performed. In this case, a separate fluorescent material may be added to the target sample solution, which causes an optical signal that can be measured and analyzed by emitting light with a specific wavelength depending on the generation of the PCR product. The light source 150c is disposed in a space between the first heater 110c and the second heater 120c and can emit the same light. The light source 150c may be operably connected to a lens (not shown) that collects light emitted from the light source 150c and an optical filter (not shown) that filters light of a specific wavelength band.

Referring to FIG. 5G, it can be confirmed that the nucleic acid amplification reaction is measured in real-time by the PCR apparatus according to the fifth embodiment of the present invention. For example, the target sample solution is passed through the reaction channel 14 sequentially through the upper-side corresponding portion 301c of the first heater 110c and the upper-side corresponding portion 302c of the second heater 120c, The target sample solution is transferred between the first heater 110c and the second heater 120c and between the first heater 110c and the second heater 120c The light source 150c passes through the corresponding upper portion of the light source 150c. When the target sample solution passes through the upper portion of the light source 150c, the flow rate of the target sample solution is slowed or temporarily held in a stopped state through fluid control, and light is emitted from the light source 150c, The light is passed through the optically transparent PCR chip 10, specifically the reaction channel 14, and the optical signal generated by nucleic acid amplification in the reaction channel 14 is measured and analyzed by the photodetector 600c can do. Therefore, by monitoring the reaction result by amplification of the nucleic acid (the fluorescent material is bound) in the reaction channel 14 during real-time during the course of each PCR cycle, the amount of the target nucleic acid can be measured in real time time can be measured and analyzed.

5H shows a PCR device according to a fifth embodiment of the present invention including a heat block of a light-transmitting material and a PCR chip, a power supply, a pump, a light supplier and an optical detector.

5H, the PCR apparatus according to the fifth embodiment of the present invention includes a thermal block 100c of a light transmitting material, a PCR chip 10, a power supply unit 400c, a pump 500c, a light supplier 700c, And an optical detector 800c. The power supply unit 400c and the pump 500c have already been described.

The PCR device according to FIG. 5H is characterized in that the thermal block 100c and the PCR chip 10 are implemented with a light-transmitting material. The PCR apparatus further includes an optical detector 700c disposed to provide light to the PCR chip 10 and an optical detector 800c disposed to receive light emitted from the PCR chip 10 . Therefore, the PCR apparatus according to the fifth embodiment of the present invention can measure and analyze the nucleic acid amplification process in real-time during PCR. In this case, a separate fluorescent material may be added to the target sample solution, which causes an optical signal that can be measured and analyzed by emitting light with a specific wavelength depending on the generation of the PCR product.

In the real-time PCR, it is necessary to increase the sensing efficiency of the optical signal as much as possible in order to accurately monitor the PCR product in real-time. Since the heat block 100c of the light transmitting material has light transmittance as a whole, most of the excitation light derived from the light source can be transmitted as it is, thereby enhancing the sensing efficiency of the optical signal. However, a part of the excitation light may be reflected on the heat block 100 of the light transmissive material or may be reflected after passing through the heat block 100c of the light transmissive material to act as noise of the optical signal. Therefore, preferably, the light absorbing material is treated on the lower surface of the heat block 100c of the light transmitting material to form a light absorbing layer (not shown), thereby further enhancing the sensing efficiency. The light absorbing substance may be, for example, mica, but is not limited as long as it is a substance having a property of absorbing light. Therefore, the light absorbing layer absorbs a part of the light derived from the light source, and the generation of the reflected light acting as noise of the optical signal can be suppressed as much as possible. Alternatively, a light reflection preventing material (not shown) may be formed on the upper surface of the heat block 100c of the light transmitting material to further enhance the sensing efficiency. 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 heat block 100c of the light transmitting material, and the light reflection preventing material is treated on the upper surface of the heat block 100c of the light transmitting material, The efficiency can be further increased. That is, in order to monitor an effective real-time PCR, the ratio of the optical signal to the noise should be as high as possible, and the ratio of the optical signal to the noise can be improved as the reflectance of excitation light from the PCR chip is low. For example, the reflectance of the excitation light of conventional heaters of a typical metallic material is about 20% to 80%, but when the heat block 100c made of a light-transmitting material including the light absorbing layer or the light reflection preventing layer is used, % To 4%, and when the heat block 100c made of a light transmitting material including the light absorbing layer and the light reflection preventing layer is used, the light reflectance can be reduced to 0.2% or less.

The light providing part 700c is a module for providing light to the PCR chip 10 of the light transmitting material and the light detecting part 800c receives the light emitted from the PCR chip 10 of the light transmitting material. And measuring the progress of the PCR performed in the PCR chip 10 of the light-transmitting material. Light is emitted from the light supplier 700c and the emitted light passes through or is reflected by the light transmitting polymer chip 10, specifically, the reaction channel 14. In this case, the reaction channel 14 The optical detecting unit 800c measures and analyzes the optical signal generated by the nucleic acid amplification in the optical detector 800c. Therefore, the reaction result by the amplification of the nucleic acid (the fluorescent substance is bound) in the reaction channel 14 during each cycle of the PCR in the light-transmissive PCR chip 10 is real- time), it is possible to measure and analyze the amplification of the target nucleic acid and the degree of amplification in real-time. In addition, the optical distributor 700c and the optical detector 800c may be disposed on the top or bottom of the column 100c of the light transmitting material, or may be disposed on the bottom of the column block 100c. However, the arrangement of the optical coupler 700c and the optical detector 800c may be varied in consideration of the arrangement relationship with the other modules in order to optimally implement the PCR apparatus. Preferably, the optical coupler 700c and the optical coupler The light detecting unit 800c may be disposed above the heat block 100c of the light transmitting material. The light supplier 700c may include a light emitting diode (LED) light source or a laser light source, a first light filter for selecting light having a predetermined wavelength from the light emitted from the light source, And a first aspheric lens disposed to diffuse the light between the light source and the first optical filter. The light source includes all light sources capable of emitting light, and according to an embodiment of the present invention, includes a light emitting diode (LED) light source or a laser light source. The first optical filter selectively emits light of a specific wavelength among incident light having various wavelength ranges and may be variously selected according to the predetermined light source. For example, the first optical filter can pass only light having a wavelength of 500 nm or less among the light emitted from the light source. The first optical lens collects the incident light and increases the intensity of the emitted light. The intensity of the light irradiated to the PCR chip 10 of the light transmitting material through the heat block 100c of the light transmitting material Can be increased. The light providing portion may further include a first aspherical lens disposed to spread light between the light source and the first optical filter. By adjusting the arrangement direction of the first aspherical lens, the light range emitted from the light source is expanded to reach the measurable area.

The photodetector 800c includes a second optical lens for collecting light emitted from the light-transmissive PCR chip 10, a second optical lens for selecting light having a predetermined wavelength in the light emitted from the second optical lens, And a light analyzer for detecting an optical signal from light emitted from the second light filter, wherein the light analyzer is arranged to collect light emitted from the second light filter between the second light filter and the light analyzer Further comprising a second aspherical lens and configured to remove noise of light emitted from the second aspherical lens between the second aspherical lens and the optical analyzer and to amplify light emitted from the second aspherical lens, And may further include a photodiode integrated circuit (PDIC) (not shown). The second optical lens collects the incident light and increases the intensity of the emitted light. The second optical lens reflects the light emitted from the PCR chip 10 of the light transmitting material through the heat block 100c of the light transmitting material Thereby increasing the intensity and facilitating optical signal detection. The second light filter selectively emits light of a specific wavelength among incident light having various wavelength bands, and is configured to emit light of predetermined wavelengths emitted from the light-transmissive material PCR chip 10 through the heat block 100c of the light- And can be variously selected depending on the wavelength of light. For example, the second optical filter can pass only light of a wavelength range of 500 nm or less of the predetermined light emitted from the light-transmissive material of the PCR chip 10 through the light block 100 of the light transmitting material . The optical analyzer is a module for detecting an optical signal from light emitted from the second optical filter. The optical analyzer converts the light emitted from the target sample solution into an electrical signal to enable qualitative and quantitative measurement. In addition, the optical detector 800c may further include a second aspherical lens disposed between the second optical filter and the optical analyzer to collect light emitted from the second optical filter. By adjusting the arrangement direction of the second aspherical lens, the light range emitted from the second optical filter is expanded to reach the measurable area. The photodetector 800c removes noise of light emitted from the second aspherical lens between the second aspherical lens and the optical analyzer, and amplifies the light emitted from the second aspherical lens. And a photodiode integrated circuit (PDIC). By using the photodiode integrated device, it is possible to miniaturize the PCR apparatus further, to minimize noise and to measure a reliable optical signal.

The PCR apparatus includes at least one dichroic filter 750x for adjusting the traveling direction of light so as to allow the light emitted from the optical supplier 700c to reach the optical detector 800c and for separating light having a predetermined wavelength, , 750y). The dichroic filters 750x and 750y are modules that selectively transmit light according to wavelengths or selectively reflect the light at a controlled angle. The dichroic filter 750x is disposed at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light supplier 700c. The dichroic filter 750x selectively transmits the short wavelength component according to the wavelength and reflects the long wavelength component at a right angle To reach the PCR chip 10 of the light-transmitting material disposed on the heat block 100c of the light-transmitting material. The dichroic filter 750y is arranged at an angle of about 45 degrees with respect to the optical axis of the light reflected from the PCR chip 10 and the heat block 100c made of a light transmissive material, Transmit a short wavelength component and reflect the long wavelength component at a right angle to reach the optical detector 800c. The light reaching the photodetector 800c is converted into an electric signal by the optical analyzer to indicate the amplification and amplification of the nucleic acid.

Referring to FIG. 5h, it can be confirmed that the nucleic acid amplification reaction is measured in real-time by the PCR apparatus according to the fifth embodiment of the present invention. For example, the target sample solution is passed through the reaction channel 14 sequentially through the upper-side corresponding portion 301c of the first heater 110c and the upper-side corresponding portion 302c of the second heater 120c, Denaturation step and PCR annealing / extension step. In this case, when the target sample solution passes through an arbitrary position in the reaction channel 14, the flow rate of the target sample solution is slowed or temporarily held in a stopped state through the fluid control, It is possible to provide light at an arbitrary position in the channel 14 and to receive light emitted by the amplification reaction of the target nucleic acid in the reaction channel 14 by the photodetection unit 800c to measure and analyze the optical signal have. 5H, the optical distributor 700c and the optical detector 800c are disposed in pairs. However, in order to measure and analyze a plurality of optical signals generated at an arbitrary position in the reaction channel 14, They can be placed in multiple pairs at appropriate locations. Therefore, by monitoring the reaction result by amplification of the nucleic acid (the fluorescent material is bound) in the reaction channel 14 during real-time during the course of each PCR cycle, the amount of the target nucleic acid can be measured in real time time can be measured and analyzed.

Experimental Example  1. Food poisoning bacteria genome DNA Extraction of

Sixteen kinds of pathogenic microorganisms causing food poisoning were purchased from the Center for Microbial Conservation of Korea, the Korea BioResource Center and The global bioresource Center. After the above-described pathogenic microorganisms were cultured, the genomic DNA of the food poisoning bacteria was extracted using QiaAmp DNA purification kit (QIAGEN).

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

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

Strain name Product
you
(bp) Primer
name Sequence Listeria monocytogenes 246 IAP-F CGG ACG TTT AAC CAA GTT GCG CTA AC (SEQ ID NO: 1) IAP-R AGC TGG GAT TGC GGT AAC AGC ATT TG (SEQ ID NO: 2) Staphylococcus aureus 169 NUCA-F AGT TCG TCA AGG CTT GGC TAA AGT TG (SEQ ID NO: 3) NUCA-R GCA GCA GTG ACA CTT TTA CAA TGA GC (SEQ ID NO: 4) Shigella spp .
136 IPAH-F ACC GCC TTT CCG ATA CCG TCT C (SEQ ID NO: 5) IPAH-R CTC TCA GTG GCA TCA GCA GCA ACA G (SEQ ID NO: 6) Vibrio parahaemolyticus 155 TOXR-F CCA AAT AGT AAT TCG CTC GCT GAC CAA CA (SEQ ID NO: 7) TOXR-R AAA CCT TGC TCA CGC CAA ACA AAC TC (SEQ ID NO: 8) Bacillus cereus 157 entFM-F CGG CCG CAC AGG TTA TGT AAG TGC AGA (SEQ ID NO: 9) entFM-R TCC AGC GAT TGA AGA TGT ATC TCC ACC TG (SEQ ID NO: 10) Salmonella spp .
204 INVA-F AAT TAT CGC CAC GTT CGG GCA ATT CGT TA (SEQ ID NO: 11) INVA-R TCA ATA ATA CCG GCC TTC AAA TCG GCA TC (SEQ ID NO: 12) Yersinia enterocolitica 143 AIL-F AAC TGG GGA GTA ATA GGT TCG TTT GCT TA (SEQ ID NO: 13) AIL-R AGG CTA ACA TAT TCG TTG ATG CGG AAA GA (SEQ ID NO: 14) Clostridium perfringens 155 CPE -F ACA ATT TAA ATC CAA TGG TGT TCG AAA ATG  C (SEQ ID NO: 15) CPE -R GGG TTC CCC TAA TAT CCA ACC ATC TCC TTT  A (SEQ ID NO: 16) Campylobacter jejuni 200 HIPO -F GCA CTT CCA TGA CCA CCC CTT CCA  A (SEQ ID NO: 17) HIPO -R CCT GCT GAA GAG GGT TTG GGT GGT GC  (SEQ ID NO: 18) Enterotoxigenic  E. coli ( ETEC ) 107 LT-F AAA ACG TTC CGG AGG TCT TAT GCC CAG A (SEQ ID NO: 19) LT-R CGG TTT GTG TTC CTC TCG CGT GAT CAT A (SEQ ID NO: 20) Enterohemorrhagic  E. coli  ( EHEC ) 159 STX1-F GCA ATT CTG GGA AGC GTG GCA TTA (SEQ ID NO: 21) STX1-R CCC CAG AGT GGA TGA ATC CCA CAA (SEQ ID NO: 22) Enteropathogenic E. coli (EPEC) 238
eaeA-F GGT GGT GTC GCT GGT CAT AC (SEQ ID NO: 23) eaeA-R CCT CTG CCG TTC CAT AAT GTT GT (SEQ ID NO: 24) Enteroinvasive E. coli (EIEC) 216
Inv-F1 CGC TTG ATG GGT GGG GTA TCC TGT CTA (SEQ ID NO: 25) Inv-R1 GTC TGC TCT TGG AGT TGA GTC ACT GTC TGT (SEQ ID NO: 26) Campylobacter coli 234
ceuE-F AGG AAT CAA TGC TGT GGA TGA (SEQ ID NO: 27) ceuE-R CTT GCT AGA TAC CAG TAT TCA GGA T (SEQ ID NO: 28) Vibrio cholerae 249
ompW-F TTG CTG ACA CTA AAC ATC TGC CAC CTA CC (SEQ ID NO: 29) ompW-R ACC ACA CAG AAG CGT TGA GGA ACC A (SEQ ID NO: 30) Vibrio vulnificus 213
Vvh-F CCG CCG CAC TTA CAT TGG TCC ATT CG (SEQ ID NO: 31) Vvh-R CCA CTG ACT TCG CCA CCC ACT TTC G (SEQ ID NO: 32) Enterohemorrhagic  E. coli  ( EHEC ) 108 STX2-F TTC CAT GAC AAC GGA CAG CAG TTA TAC CA (SEQ ID NO: 33) STX2-R AAC GCC AGA TAT GAT GAA ACC AGT GAG TGA (SEQ ID NO: 34) Enterotoxigenic  E. coli ( ETEC ) 240 ST-F TCC GTG AAA CAA CAT GAC GGG AGG TA (SEQ ID NO: 35) ST-R CAT GGA GCA CAG GCA GGA TTA CAA CA (SEQ ID NO: 36)

Experimental Example  3. Third aspect of the present invention In the embodiment  Following PCR  Real time using device PCR  Perform

1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 3 and SEQ ID NO: 4 in the primer set for detecting food poisoning bacteria according to an embodiment of the present invention described in Table 1 using the genomic DNA of the food poisoning bacteria obtained in Experimental Example 1 as a template, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: Among the primer sets for detecting food poisoning bacteria according to one embodiment of the present invention described in Table 1, as the first group, there are provided a group consisting of SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: And SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: As the third group, 35 and SEQ ID NO: 36 were introduced into the PCR chip according to the second embodiment of the present invention, which had 10 reaction channels, respectively, into four groups. The first PCR chip comprises a negative control (No-1 reaction channel, No template), a positive control (Positive control DNA), SEQ ID NO: 1 and SEQ ID NO: 2 SEQ ID NO: 3 and SEQ ID NO: 4 (Reaction Channels 1-4), SEQ ID NO: 5 and SEQ ID NO: 6 (Reaction Channels 1-5), SEQ ID NO: 7 and SEQ ID NO: 9 and SEQ ID NO: 10 (1-7 reaction channels), SEQ ID NO: 11 and SEQ ID NO: 12 (1-8 reaction channels), SEQ ID NO: 13 and SEQ ID NO: 14 (Second-1 reaction channel, positive control DNA), a sequence of SEQ ID NO: 16 (1-10th reaction channel), and the second PCR chip comprises a negative control SEQ ID NO: 19 and SEQ ID NO: 20 (2-4 reaction channel), SEQ ID NO: 21 and SEQ ID NO: 22 (2-5 reaction channel), SEQ ID NO: 23 And SEQ ID NO: 24 (2-6 reaction channel), SEQ ID NO: 25 and SEQ ID NO: 27, SEQ ID NO: 27 and SEQ ID NO: 28 (Reaction Channels 2-8), SEQ ID NO: 29 and SEQ ID NO: 30 (Reaction Channels 2-9), SEQ ID NO: 31 and SEQ ID NO: 32 (2nd-10th reaction channel), the third PCR chip includes SEQ ID NO: 33 and SEQ ID NO: 34 (3-1 reaction channel) without the control group and the fourth PCR chip comprises SEQ ID NO: 35 and SEQ ID NO: 36 (4-1 reaction channel). Real-time PCR was performed using the PCR apparatus according to the third embodiment of the present invention based on the first to fourth PCR chips. In carrying out the real-time PCR using the PCR apparatus according to the third embodiment of the present invention, the composition of the reaction solution for the real-time PCR and the reaction conditions of the real-time PCR are shown in Tables 2 and 3 below.

Furtherance Volume ([mu] l) 2 x NBS real-time PCR master mix (SYBR Green 1) 6.5 Forward primer (10 [mu] M) 1.3 The reverse primer (10 [mu] M) 1.3 Template DNA (1 x 10 ⁶ copies) One Distilled water (D.W.) 2.9 Total (total) 13

step Temperature (℃) time Cycles Pre-denaturation 95 8 sec One Denaturation 90 3 sec 30 Annealing & Extension 72 6 sec

6A is an electrophoresis image showing a real-time PCR result of a sample using the PCR apparatus according to the third embodiment of the present invention based on the first PCR chip, FIG. 9A is an electrophoresis image showing a real-time PCR result for a sample using the PCR apparatus according to the third embodiment of the present invention. FIG. 9A is a third example of the present invention 9B is an electrophoresis image showing a result of real-time PCR on a sample using a PCR apparatus according to an example. FIG. 9B is a graph showing the electrophoresis of a sample using the PCR apparatus according to the third embodiment of the present invention, Of the present invention. In this case, the numbers shown in the electrophoresis photographs of Figs. 6A and 6B are reaction channel numbers. 6A and 6B, it can be confirmed that the food poisoning gene was accurately and rapidly detected at the same time by using the primer set and the PCR apparatus according to one embodiment of the present invention. According to FIGS. 9A and 9B, It was confirmed that the pathogen gene was accurately and rapidly detected individually using the primer set and PCR apparatus according to the examples.

FIG. 7A is a graph showing a real-time PCR result of a sample using the PCR device according to the third embodiment of the present invention based on the first PCR chip, FIG. 7B is a graph showing the result of the real- FIG. 10A is a graph showing the results of real-time PCR for a sample using the PCR apparatus according to the third embodiment of the present invention. FIG. 10A is a graph showing the results of the PCR according to the third embodiment of the present invention FIG. 10B is a graph showing the results of real-time PCR for a sample using the apparatus, and FIG. 10B is a graph showing a real-time PCR for a sample using the PCR apparatus according to the third embodiment of the present invention, Fig. 7A and 7B, it can be confirmed that the food poisoning gene was detected accurately and in real time at the same time by using the primer set and the PCR apparatus according to one embodiment of the present invention. According to FIGS. 10A and 10B, The gene of the food poisoning bacteria was detected accurately and quickly in real time using the primer set and PCR apparatus according to one embodiment of the present invention. 7A and 7B, the Ct values of the real-time PCR corresponding to the respective reaction channel numbers of the first PCR chip and the second PCR chip are shown in Figs. 8A and 8B, , The Ct value of the real time PCR is 21.06. In the real time PCR result graph of FIG. 10B, the Ct value of the real time PCR is 24.00.

<110> NANOBIOSYS Inc          KOREA FOOD & DRUG ADMINISTRATION          Korea Polytech Specialized College Industrial Cooperation Corp. <120> PCR device for detecting food-borne bacteria, and method for          detecting food-borne bacteria using the same <130> WPN13028 <160> 36 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 1 cggacgttta accaagttgc gctaac 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 2 agctgggatt gcggtaacag catttg 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 3 agttcgtcaa ggcttggcta aagttg 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 4 gcagcagtga cacttttaca atgagc 26 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 5 accgcctttc cgataccgtc tc 22 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 6 ctctcagtgg catcagcagc aacag 25 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 7 ccaaatagta attcgctcgc tgaccaaca 29 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 8 aaaccttgct cacgccaaac aaactc 26 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 9 cggccgcaca ggttatgtaa gtgcaga 27 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 10 tccagcgatt gaagatgtat ctccacctg 29 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 11 aattatcgcc acgttcgggc aattcgtta 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 12 tcaataatac cggccttcaa atcggcatc 29 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 13 aactggggag taataggttc gtttgctta 29 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 14 aggctaacat attcgttgat gcggaaaga 29 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 15 acaatttaaa tccaatggtg ttcgaaaatg c 31 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 16 gggttcccct aatatccaac catctccttt a 31 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 17 gcacttccat gaccacccct tccaa 25 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 18 cctgctgaag agggtttggg tggtgc 26 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 19 aaaacgttcc ggaggtctta tgcccaga 28 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 20 cggtttgtgt tcctctcgcg tgatcata 28 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 21 gcaattctgg gaagcgtggc atta 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 22 ccccagagtg gatgaatccc acaa 24 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 23 ggtggtgtcg ctggtcatac 20 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 24 cctctgccgt tccataatgt tgt 23 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 25 cgcttgatgg gtggggtatc ctgtcta 27 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 26 gtctgctctt ggagttgagt cactgtctgt 30 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 27 aggaatcaat gctgtggatg a 21 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 28 cttgctagat accagtattc aggat 25 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 29 ttgctgacac taaacatctg ccacctacc 29 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 30 accacacaga agcgttgagg aacca 25 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 31 ccgccgcact tacattggtc cattcg 26 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 32 ccactgactt cgccacccac tttcg 25 <210> 33 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 33 ttccatgaca acggacagca gttatacca 29 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 34 aacgccagat atgatgaaac cagtgagtga 30 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 35 tccgtgaaac aacatgacgg gaggta 26 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 36 catggagcac aggcaggatt acaaca 26

Claims (23)

delete delete delete delete delete delete delete delete A light-transmitting layer disposed on the substrate, a heat-generating layer including conductive nanoparticles disposed on the substrate, an insulating protective layer disposed on the heat-generating layer, and electrodes connected to the heat-generating layer, ; And
And a PCR chip disposed so as to be able to contact the upper surface of the light-transmitting heat block,
The PCR chip comprises: a first plate; A second plate disposed on the first plate and having at least one reaction channel; And a third plate disposed on the second plate and having an inlet and an outlet connected to both ends of the at least one reaction channel, the inlet and the outlet being configured to be openable and closable, wherein different food poisoning bacteria Comprising a primer set for detection,
The primer set includes:
Detecting an iap (invasion associated protein) gene of Listeria monocytogenes consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2 Primer set;
A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer for detecting the nucA (nuclease) gene of Staphylococcus aureus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: set;
A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 6 . A primer set for detecting an ipaH (invasive plasmid antigen gene) gene;
A primer set for detecting a toxR gene of Vibrio parahaemolyticus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 8;
A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 9 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 10, for detecting the enterotoxin FM (entFM) gene of Bacillus cereus Primer set;
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 . A primer set for detecting an invA protein of an invA ;
Detecting an ail (attachment invasion locus) gene of Yersinia enterocolitica consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 14 Primer set;
Detecting the cpe (C. perfringens enterotoxin) gene of Clostridium perfringens comprising a primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the A primer set;
A primer set for detecting a hypo gene of Campylobacter jejuni consisting of a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 17 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 18;
( LT ) of Enterotoxigenic E. coli (ETEC) 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 a primer set for detecting an enterotoxin gene;
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 , the STX1 (shiga-like ) of Enterohemorrhagic E. coli (EHEC) a primer set for detecting a toxin-1 gene;
Detecting the eaeA gene of Enteropathogenic E. coli (EPEC) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set;
Detecting the invA gene of Enteroinvasive E. coli (EIEC) consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 25 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 26 Primer set;
A primer set for detecting a ceuE gene of Camphylobacter coli comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 27 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 28;
A primer set for detecting an ompW gene of Vibrio cholera consisting of 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 set for detecting a Vvh gene of Vibrio vulnificus comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 31 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 32;
A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 33 and a STX2 (shiga-like ) gene of Enterohemorhhagic E. coli (EHEC) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: a primer set for detecting a toxin-2 gene; And
( ST ) of Enterotoxigenic E. coli (ETEC) consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 35 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: and a primer set for detecting an enterotoxin gene. 10. The method of claim 9,
The conductive nanoparticles included in the heating layer may be doped with an impurity selected from the group consisting of In, Sb, Al, Ga, C, and Sn to an oxide semiconductor material or the oxide semiconductor material. Wherein the insulating protective layer is selected from the group consisting of dielectric oxides, perylene, nanoparticles, and polymer films, and wherein the electrode is selected from the group consisting of a metal material, a conductive epoxy, a conductive paste, a solder, and a conductive film Characterized by a PCR device. 10. The method of claim 9,
Wherein a light absorbing layer containing a light absorbing material is disposed in contact with the lower surface of the substrate of the light transmitting thermal block or a light reflecting layer having a light reflecting preventive material is disposed in contact with the upper surface of the insulating protective layer of the light transmitting thermal block Wherein the PCR product is a PCR product. 10. The method of claim 9,
The PCR apparatus further comprises an optical detector disposed to be drivable to provide light to the PCR chip disposed on the chip contact portion and a photodetecting portion driveably arranged to receive light emitted from the PCR chip disposed on the chip contact portion And a PCR device. A heater unit including two heater groups each having one or more heaters and arranged so that two heater groups are spaced apart from each other so as to prevent mutual heat exchange, A thermal block having a contact surface;
An electrode unit having an electrode connected to supply electric power to the heaters provided in the column block; And
And a PCR chip disposed so as to be able to be in contact with the thermal block so as to be capable of heat exchange with one or more heaters provided in the thermal block,
Wherein the two heater groups include a first heater group and a second heater group, wherein the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, The first heater group maintains the PCR annealing / extension step temperature, the second heater group maintains the PCR denaturation step temperature,
The PCR chip comprises: a first plate; A second plate disposed on the first plate and having at least one reaction channel; And a third plate disposed on the second plate and having an inlet and an outlet connected to both ends of the at least one reaction channel, the inlet and the outlet being configured to be openable and closable, wherein different food poisoning bacteria Comprising a primer set for detection,
The primer set includes:
Detecting an iap (invasion associated protein) gene of Listeria monocytogenes consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2 Primer set;
A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer for detecting the nucA (nuclease) gene of Staphylococcus aureus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: set;
A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 6 . A primer set for detecting an ipaH (invasive plasmid antigen gene) gene;
A primer set for detecting a toxR gene of Vibrio parahaemolyticus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 8;
A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 9 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 10, for detecting the enterotoxin FM (entFM) gene of Bacillus cereus Primer set;
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 . A primer set for detecting an invA protein of an invA ;
Detecting an ail (attachment invasion locus) gene of Yersinia enterocolitica consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 14 Primer set;
Detecting the cpe (C. perfringens enterotoxin) gene of Clostridium perfringens comprising a primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the A primer set;
A primer set for detecting a hypo gene of Campylobacter jejuni consisting of a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 17 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 18;
( LT ) of Enterotoxigenic E. coli (ETEC) 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 a primer set for detecting an enterotoxin gene;
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 , the STX1 (shiga-like ) of Enterohemorrhagic E. coli (EHEC) a primer set for detecting a toxin-1 gene;
Detecting the eaeA gene of Enteropathogenic E. coli (EPEC) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set;
Detecting the invA gene of Enteroinvasive E. coli (EIEC) consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 25 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 26 Primer set;
A primer set for detecting a ceuE gene of Camphylobacter coli comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 27 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 28;
A primer set for detecting an ompW gene of Vibrio cholera consisting of 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 set for detecting a Vvh gene of Vibrio vulnificus comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 31 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 32;
A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 33 and a STX2 (shiga-like ) gene of Enterohemorhhagic E. coli (EHEC) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: a primer set for detecting a toxin-2 gene; And
( ST ) of Enterotoxigenic E. coli (ETEC) consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 35 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: and a primer set for detecting an enterotoxin gene. delete delete delete 14. The method of claim 13,
Wherein the thermal block is implemented to have optical transparency. 14. The method of claim 13,
Wherein the heater provided in the thermal block includes a light-transmitting heat generating element. 14. The method of claim 13,
Further comprising: a power supply for supplying power to the electrode unit; and a pump arranged to provide positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the at least one reaction channel. 14. The method of claim 13,
A light source is disposed between the first heater and the second heater and a power supply for supplying electric power to the electrode unit; a positive pressure or negative pressure for controlling a flow rate and a flow rate of the fluid flowing in the at least one reaction channel; And a light detector for detecting the light emitted from the light source. 14. The method of claim 13,
A pump arranged to provide positive or negative pressure to control the flow rate and flow rate of fluid flowing in the at least one reaction channel, a pump arranged to provide light to the PCR chip, And a photodetector part arranged to receive light emitted from the PCR chip and light emitted from the PCR chip. A heater unit including three heater groups each having one or more heaters, wherein the heater units are repeatedly arranged so as to prevent mutual heat exchange between the heater groups, and wherein at least one surface of the heater unit is provided with a PCR chip A thermal block having a contact surface;
An electrode unit having an electrode connected to supply electric power to the heaters provided in the column block; And
And a PCR chip disposed so as to be able to be in contact with the thermal block so as to be capable of heat exchange with one or more heaters provided in the thermal block,
Wherein the three heater groups include a first heater group, a second heater group and a third heater group, wherein the first heater group maintains the PCR denaturation temperature and the second heater group maintains the PCR annealing temperature Wherein the third heater group maintains the PCR extension step temperature or the first heater group maintains the PCR annealing step temperature and the second heater group maintains the PCR extension step temperature and the third heater group performs the PCR denaturation step Or the first heater group maintains the PCR extension step temperature, the second heater group maintains the PCR denaturation step temperature, the third heater group maintains the PCR annealing step temperature,
The PCR chip comprises: a first plate; A second plate disposed on the first plate and having at least one reaction channel; And a third plate disposed on the second plate and having an inlet and an outlet connected to both ends of the at least one reaction channel, the inlet and the outlet being configured to be openable and closable, wherein different food poisoning bacteria Comprising a primer set for detection,
The primer set includes:
Detecting an iap (invasion associated protein) gene of Listeria monocytogenes consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2 Primer set;
A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 and a primer for detecting the nucA (nuclease) gene of Staphylococcus aureus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: set;
A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 6 . A primer set for detecting an ipaH (invasive plasmid antigen gene) gene;
A primer set for detecting a toxR gene of Vibrio parahaemolyticus comprising a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 8;
A primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 9 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 10, for detecting the enterotoxin FM (entFM) gene of Bacillus cereus Primer set;
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 . A primer set for detecting an invA protein of an invA ;
Detecting an ail (attachment invasion locus) gene of Yersinia enterocolitica consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 14 Primer set;
Detecting the cpe (C. perfringens enterotoxin) gene of Clostridium perfringens comprising a primer comprising SEQ ID NO: 15 of the nucleotide sequence of a primer comprising at least 15 consecutive nucleotides, and SEQ ID NO: 16 nucleotide sequence at least 15 consecutive nucleotides of the A primer set;
A primer set for detecting a hypo gene of Campylobacter jejuni consisting of a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 17 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 18;
( LT ) of Enterotoxigenic E. coli (ETEC) 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 a primer set for detecting an enterotoxin gene;
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 , the STX1 (shiga-like ) of Enterohemorrhagic E. coli (EHEC) a primer set for detecting a toxin-1 gene;
Detecting the eaeA gene of Enteropathogenic E. coli (EPEC) comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 24 Primer set;
Detecting the invA gene of Enteroinvasive E. coli (EIEC) consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 25 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 26 Primer set;
A primer set for detecting a ceuE gene of Camphylobacter coli comprising a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 27 and a primer comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 28;
A primer set for detecting an ompW gene of Vibrio cholera consisting of 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 set for detecting a Vvh gene of Vibrio vulnificus comprising a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 31 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 32;
A primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 33 and a STX2 (shiga-like ) gene of Enterohemorhhagic E. coli (EHEC) consisting of a primer comprising 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: a primer set for detecting a toxin-2 gene; And
( ST ) of Enterotoxigenic E. coli (ETEC) consisting of a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 35 and a primer comprising at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: and a primer set for detecting an enterotoxin gene. Introducing a sample suspected of causing foodborne pathogens into a PCR apparatus according to any one of claims 9 to 13 and 17 to 22 to perform PCR; And
And confirming the presence or absence of food poisoning bacteria in the target sample from the PCR result.

Images