KR102114148B1 - Method for Detecting Foodborne Microorganism - Google Patents

Abstract

According to a specific example of the present disclosure, it detects microorganisms in food in a short time with good reliability compared to a pathogen detection method such as a conventional food poisoning bacteria that requires a complicated procedure over a long period of time from food (specifically, instant food sold in convenience stores). New methods are described.

Description

Method for detecting microorganisms from food {Method for Detecting Foodborne Microorganism}

The present disclosure relates to a method for detecting microorganisms from food. More specifically, the present disclosure detects microorganisms in food with good reliability in a short period of time compared to conventional methods for detecting pathogens such as food poisoning bacteria that require complicated procedures over a long period of time from food (specifically, instant food sold in convenience stores). It relates to a novel method.

In the intestines of all warm-blooded animals, including humans, a variety of pathogens such as bacteria and viruses are inhabited, providing a very good environment. Pathogens are widely distributed in the surrounding environment, and specifically, bacterial pathogens are found in soil, animal organs, and water contaminated by animal stools. The human body, on average, has more than 150 types of bacteria both inside and outside the body, although many microorganisms are harmless to the human body, some of which are food poisoning, cholera, diarrhea, and vomiting. It causes various infectious diseases including pneumonia and typhoid. In particular, over 200 types of disease can be transmitted through food and drinking water alone. For example , the Escherichia Coli (or E.coli ) family does not cause major problems when ingested, but some of them are known to produce toxins that cause diarrhea and the like.

In particular, E. coli O157: H7, a pathogenic E. coli , is an intestinal hemorrhagic E. coli, widely known as a causative agent of food poisoning, and is a problem worldwide. In addition, infection causes hemolytic uremic syndrome, hemorrhagic colitis, diarrhea, kidney failure, and seizures, leading to death in severe cases (Reisner, A. et al. , 2006. Journal of Bacteriology. 188, 3572-3581) ; Barrientos, RM et al. , 2009. Brain, Behavior, and Immunity. 23, 450-454; Schrag, SJ et al. , 2006. PEDIATRICS. 118, 570-576). Salmonella choleraesuis , Salmonella bongori , and Salmonella typhimurium are also representative pathogens that cause food poisoning, and are the causative agents of typhoid and paratyphoid, and can be contaminated with various livestock and animals. In addition, if proper washing, processing temperature and storage conditions are not observed, Salmonella can multiply and cross-contaminated with other foods through contaminated drinking water and food.

On the other hand, pathogens, especially food poisoning bacteria, can be easily infected by humans through contaminated foods in general. Under normal conditions, the rate of reproduction of pathogens is very fast, so even if a small number of pathogens invade the human body once, they grow rapidly in the intestine, which is very suitable for their growth environment, to a level that can threaten human health. Therefore, there is a need for a diagnostic technique that can quickly and easily detect the presence or absence of a pathogen (especially E. coli ) from a contaminated environment.

In particular, in the case of ready-to-eat foods sold in convenience stores, etc., since there is no clear heating process to destroy microorganisms before consumption by consumers, serious food poisoning can be caused when contaminated with bacteria. Therefore, the process of setting and detecting hygiene standards such as harmful microorganisms for instant food is essential for preventing food poisoning and food accidents by consumers. In particular, with the expansion of the instant food market, there is an increasing need for the development of a technology for detecting microorganisms capable of causing food poisoning in foods because food companies conduct a total inspection to prevent food poisoning accidents that may occur in the food manufacturing process.

The conventional method for diagnosing or monitoring food poisoning bacteria contained in instant food, food provided in a restaurant, etc. is a method for quantitative analysis by cultivating bacteria to increase the number, and the characteristics of the medium grown for each bacteria are different, and thus the bacteria are used. Although it has been identified, this method has a disadvantage in that a long time of about 3 to 5 days is required through a complicated process by an expert trained in a laboratory equipped with equipment to obtain a final result. In addition, detection of similar species in food poisoning bacteria may cause test confusion, and it is practically impossible to detect food poisoning bacteria in the field.

On the other hand, for the diagnosis of environmental samples containing pathogens such as E.coli , ATP measurement method using the principle of luminescence by reaction of ATP (adenosine triphosphate) and luciferin / luciferase has been recently applied. .

In the ATP measurement method, luciferin, a light-emitting substance, is activated by reacting with ATP, and is activated by a luminase, luciferase, oxidized by oxygen to convert chemical energy into light energy and emit light. That is, luciferin, a luminescent material, combines with ATP to form a complex of luciferin-ATP, producing two inorganic phosphoric acid (H ₃ PO ₄ ) molecules, wherein luciferin is a reduced form. The reduced luciferin-AMP generated in the reaction is in an unstable energy state as it is oxidized by reacting with oxygen, so that the unstable oxidation product is rapidly decomposed to emit light ( hv ) while producing oxidized luciferin and AMP. do. At this time, the amount of emitted light can be measured and quantified. ATP is an indicator of the existence of living things. In other words, if microorganisms are present, ATP, which is essentially contained in cells, is also present, and thus can emit light.

The above-described method has the advantage of detecting not only microorganisms but also other organic substances, but since a small amount of ATP is contained in a sample such as food, ATP derived from food, etc. is removed for accurate diagnosis, and the target of detection is Only microbial-derived ATP should be measured. As such, the APT measurement method itself has a fundamental limitation in collecting only harmful microorganisms and performing an accurate diagnosis. In particular, when the concentration of the target pathogen to be diagnosed in the sample is low, the difficulty of accurate detection and diagnosis may be exacerbated.

As alternative methods for diagnosing the presence or absence of a pathogen in a sample are continuously being studied, nucleic acid-based detection methods such as DNA and RNA are used. Among the above methods, in the case of nucleic acids, especially DNA, it is widely used because it can increase the diagnostic sensitivity by a powerful amplification technique called polymerase chain reaction (PCR). In general, PCR is a molecular biological method for amplifying DNA gene samples in vitro, and is a technique of increasing the amount and concentration of DNA samples to increase sensitivity to DNA. This PCR technique is a chemical reaction in which temperature control is very important, and is a method of repeatedly heating and cooling a target sample in three temperature profiles (typically 55-65 ° C, 72 ° C, and 95 ° C, respectively). However, even in the case of using the PCR method, when external DNA other than the DNA of the bacteria is present in the sample, when PCR is performed using these as a template, there is a high risk of false positives that may have completely different results.

Therefore, it solves the problems of the prior art, and more quickly (e.g., within about 4 hours), a reliable detection method, especially for samples of foods, especially ready-to-eat foods, in which pathogens such as food poisoning bacteria are present at low concentration It is urgently required.

In an embodiment according to the present disclosure, in order to overcome the limitations of the prior art, it is intended to provide a method for detecting microorganisms (specifically pathogens such as food poisoning bacteria) in food by performing a specific pre-treatment process.

In addition, in the embodiment according to the present disclosure, it is intended to provide a reliable detection method within a short time of a few hours, unlike the conventional method, which takes several days to detect the pathogen in the food sample and has a low detection limit.

According to an embodiment of the present disclosure,

a) preparing a sample-containing buffer solution by injecting a buffer solution into the food sample;

b) centrifuging the sample-containing buffer solution under conditions of a rotational speed of 100 to 5000 rpm to separate into pellets and supernatant containing food samples in the form of particulates;

c) pre-treating the supernatant through a membrane filter;

d) dissolving the retentate of the membrane filter to extract a gene from the retainer;

e) amplifying the extracted gene; And

f) quantitatively, qualitatively or quantitatively / quantitatively detecting microorganisms from the amplification products produced as a result of step e);

Provided is a method for detecting microorganisms from foods comprising the.

According to an exemplary embodiment, the food product may be rice or bakery.

According to an exemplary embodiment, prior to step b), the method may further include reducing the size of the sample in the sample-containing buffer solution.

According to an exemplary embodiment, the membrane filter,

An upper substrate on which an injection hole of the supernatant is formed;

A filter structure positioned under the upper substrate and having a membrane interposed between the first and second intermediate support substrates having a mesh structure; And

A lower substrate positioned under the filter structure and having a discharge channel for discharging the filtrate filtered by the filter structure;

It may include.

According to an exemplary embodiment, a first guide substrate having a channel and a cavity for guiding the supernatant injected from the upper substrate to the filter structure is interposed between the upper substrate and the filter structure,

A second guide substrate having a channel and a cavity for guiding the filtrate to the lower substrate may be interposed between the filter structure and the lower substrate.

According to an exemplary embodiment, a cavity having a shape conforming to a membrane is formed between each of the first intermediate supporting substrate and the membrane, and between the membrane and the second intermediate supporting substrate to receive and fix the membrane in combination. And a fourth intermediate support substrate.

 According to an exemplary embodiment, step e) may be performed using a microfluidic gene amplification device.

According to an exemplary embodiment, the microfluidic gene amplification device,

(i) a gene amplification unit having at least one microchannel type gene amplification chamber;

(ii) a heating member disposed in contact with or adjacent to the gene amplification unit to supply heat energy necessary for a gene amplification reaction; And

(iii) a control unit for controlling the gene amplification reaction;

It may include.

According to an exemplary embodiment, the gene amplification unit is an upper side open at least one trench (trench) or groove (groove) is formed in the lower sheet; And a chamber for gene amplification formed by covering at least one trench or groove that is bonded to the lower sheet and the upper side is open, wherein the chamber for gene amplification is a space for receiving and gene amplifying a fluid containing the extracted gene. Can provide

According to an exemplary embodiment, the gene amplification unit may further include a microvalve for controlling inflow and outflow of fluid introduced into the microchannel.

A method for detecting microorganisms from food provided according to an embodiment of the present disclosure, specifically, instant food, overcomes the fact that it takes several days to detect pathogens in food samples in the past, and is reliable even within a short period of time. Detection results can be provided.

In particular, as it can be effectively applied to samples containing low concentrations of microorganisms (for example, pathogens such as food poisoning bacteria), it is applicable as a field test technique.

1 is a flow chart showing a series of methods for detecting or diagnosing microorganisms from ready-to-eat foods according to one embodiment;
2 is a view schematically showing the structure of a membrane filter for separating a supernatant obtained by centrifugation into a retainer and a filtrate (filtrate) in an exemplary embodiment;
3 is a microfluidic device for gene amplification in an exemplary embodiment, schematically showing a gene amplification unit in a gene amplification apparatus;
4 shows the results of electrophoresis analysis of amplification products obtained through PCR while changing the concentration of bacteria in the sample extracted from the instant food; And
5 is a standard filter of the gene amplification product obtained through RT-PCR while changing the concentration of bacteria in the sample, and the supernatant obtained from the centrifugation performed in the range of 500 to 2000 rpm in the Example membrane filter It is a graph showing the results of the gene amplification test obtained through RT-PCR while changing the concentration of bacteria in the sample extracted through.

The present invention can be achieved by the following description with reference to the accompanying drawings. It should be understood that the following description describes preferred embodiments of the invention, and it is to be understood that the invention is not necessarily limited thereto.

In addition, the accompanying drawings may be expressed somewhat exaggerated compared to the thickness (or height) of the actual layer or a ratio with other layers in order to facilitate understanding, and its meaning will be properly understood by the specific purpose of the related description to be described later. Can be.

Terms used in the present specification may be defined as follows.

“Instant food” means any food that has been fully or partially cooked, and is typically sold in a retail market, such as a convenience store, and can be consumed on the fly or by simple processing such as heating. It can be understood as.

"Sample" is not limited to a specific type or form, as long as it can contain the microorganism to be detected, but for the purpose of this specification, it mainly refers to a type derived from a carbohydrate food having a texture texture such as rice, bakery, etc. Can mean

"Microorganism" in a broad sense may mean a substance derived from any type of biological organism, and may specifically consist of a plurality of cells, for example, pathogens (pathogenic cells, bacteria, etc.). have.

"Bacteria", the outer membrane (bacterial envelope) of the Gram (Gram) staining reaction is distinguished according to the structure, the Gram-negative bacteria (thin murine (murein) or peptidoglycan layer and has a lipid bilayer of the outer membrane) and Gram-positive bacteria (thick murine or peptidoglycan layer with crystal violet). The cell membrane of gram-negative bacteria consists of phospholipids and other glycoproteins, and most of the phospholipids have a negative charge (net negative charge). The types include Salmonella, meningitis, spiroheta cholera, plague, typhoid, heterogeneous, E. coli, gonorrhea. On the other hand, in the case of Gram-positive bacteria, the cell wall is mainly composed of peptidoglycan and teichoic acid, and its surface has almost neutral charge characteristics, and staphylococcus, streptococcus, and anthrax , Diphtheria, tetanus, and pneumonia.

“Nucleic acid” may also refer to a plurality of nucleotides consisting of sugars linked to organic bases (substituted pyrimidines such as cytosine, thymidine or uracil or substituted purines such as aden or guanine). That is, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), methylphosphonate nucleic acid, S-oligo, c-DNA, cRNA, miRNA, siRNA, aptamer, etc. can be exemplified, and naturally occurring Or it may be a concept that includes both artificially synthesized. However, more typically, it may be DNA, RNA, and the like.

“Amplification” may mean forming a plurality of copies of a nucleic acid molecule or a plurality of complementary complements to a nucleic acid molecule by using at least one of the nucleic acid molecules as a template, such an amplification system or Amplification means include, for example, PCR systems, ligase chain reaction (LCR) systems, nucleic acid sequence based amplification (NASBA), Q-Beta Replicase systems, transcription-based amplification systems (TAS) And strand displacement amplification (SDA), and the like, and amplification products are referred to as amplicons.

“Buffer” is a pKa of about 6 to about 9 at 25 ° C., for example, and a pH value of 6 to 9 (specifically about 7.5 to 9, more specifically about 7.8 to 8.5). It may mean a composition that can effectively maintain. At this time, the buffer solution may mean a physiologically compatible buffer solution that is typically harmonized with the function of enzyme activity and maintains normal physiological and biochemical functions of biological molecules.

“Cell lysis or lysis” may mean a phenomenon in which cell membranes are ruptured and cell contents are exposed according to cell decomposition, and DNA or RNA is usually extracted in the entire stage of an amplification process such as PCR or It is used a lot to separate.

"PCR" refers to a reaction in which a large amount of identical DNA strands are formed from one original template by a cycle process (heating and cooling are alternately performed). Typically, PCR mixtures are (i) sequencing sequences to be amplified. A double-stranded DNA molecule, (ii) a primer (a single-stranded DNA molecule capable of binding to a complementary DNA sequence in the template DNA), (iii) a mixture of dATP, dTTP, dGTP, and dCTP (in the PCR amplification process) Nucleotide subunits) that are joined to form a new DNA molecule) dNTP, and (iv) an enzyme that synthesizes a new DNA molecule using Taq DNA polymerase (dNTP)).

“Microfluidic channel” means a channel with a largest dimension typically in the range of 1000 μm or less, specifically about 1 to 500 μm, and more specifically about 10 to 300 μm in the cross-sectional area of the elongated passageway through which the fluid flows. can do.

"Intercalating dye" may mean a dye having the property of binding to a major groove of a nucleic acid, particularly a DNA double strand.

The term "fluorescence" may refer to a type of emission generated for a short time by electromagnetic excitation. That is, fluorescence refers to a phenomenon generated when a specific substance absorbs light energy at a short wavelength and then emits light energy at a longer wavelength. Illustratively, the length of time between absorption and release is typically relatively short, for example on the order of about 10 ^-9 to 10 ^-8 seconds.

The “Ct value” can be defined as the number of cycles required for a fluorescence signal exceeding a threshold (a value exceeding the background level) in real time PCR, inversely proportional to the amount of target nucleic acid in the sample (ie, the Ct value is The smaller the larger the amount of target nucleic acid in the sample).

The expressions “on” and “on” can be understood to be used to refer to the concept of relative position. Thus, as well as when other components or layers are present directly in the recited layer, other layers (intermediate layers) or components may be interposed or present therebetween. Similarly, the expressions “below”, “below” and “below” and “between” may also be understood as relative concepts of location. In addition, the expression "sequentially" can also be understood as a concept of relative position.

1 is a flowchart showing a series of methods for detecting or diagnosing microorganisms from ready-to-eat foods according to an embodiment of the present disclosure, and detailed steps will be described with reference to the drawings.

Preparation of food sample-containing buffer solution

According to one embodiment of the present disclosure, the target of detection or diagnosis may be a type of food, specifically, an instant food, which may be a type that requires a separate treatment due to difficulty in accurate diagnosis or detection as it is being sold. Examples include rices such as gimbap, bakers such as sandwich, and the like.

According to one embodiment, a sample-containing buffer solution is prepared by injecting a buffer solution into a food sample. A typical example of the buffer solution is PBS (Phosphate buffered saline), an aqueous salt solution mainly containing disodium hydrogen phosphate and sodium chloride. Can be The buffer solution may be added in an amount suitable for subsequent treatment (especially centrifugation), for example, about 10 to 150 parts by weight (specifically about 20 to 120 parts by weight, relative to 100 parts by weight of the food sample to be diagnosed) More specifically, about 50 to 100 parts by weight of the buffer solution may be injected. However, the injection amount range of the buffer solution can be changed according to the type of food.

However, in some cases, it may be advantageous to selectively perform a size reduction step to have an appropriate size depending on the food sample. As such an optional step, sieving, crushing (for example, wet crushing), grinding using a blender or a mixer, and the like can be exemplified, and these operations may be performed by combining two or more.

Centrifugation of food sample-containing buffer solution

After preparing the sample-containing buffer solution as described above, it can be separated into a supernatant liquid on the upper side and a pellet on the lower side through centrifugation. At this time, food samples in the form of small particles are dispersed in the supernatant.

According to an exemplary embodiment, the size of the food sample in the sample-containing buffer solution to be centrifuged may range from, for example, about 10 mesh or less, specifically about 20 to 120 mesh, and more specifically about 50 to 100 mesh. Bar, as described above, if it exceeds the appropriate size, the supernatant formed by the subsequent centrifugation may have an undesired effect on the pretreatment or detection process by the membrane due to the presence of too much or a small amount of sample particles. .

On the other hand, according to one embodiment, in the centrifugal separation process, the rotational speed needs to be adjusted within a predetermined range. That is, under excessively high rotational speed conditions, the microorganisms to be detected (specifically, pathogens) are precipitated by binding with food lumps, whereas if the rotational speed is too low, the food lumps tend not to precipitate into pellets. In consideration of the above points, in one embodiment, for example, centrifugation may be performed at a rotational speed selected from a range of about 100 to 5000 rpm, specifically about 300 to 3000 rpm, and more specifically about 500 to 2000 rpm. Can be.

In addition, the centrifugation time may range, for example, from about 20 to 50 seconds, more specifically from about 25 to 45 seconds, and more specifically from about 30 to 40 seconds, but this should be understood for illustrative purposes.

In addition, when centrifugation, the temperature may be advantageous for effective separation between pellets of food samples that are discarded to be kept in a range that is not too high and food sample particles dispersed in the supernatant. For example, it may be selected within a range of about 2 to 15 ° C, specifically about 3 to 10 ° C, and more specifically about 4 to 7 ° C.

The size of the food sample particles dispersed in the supernatant according to the above-described centrifugation is, for example, about 20 μm to 3 mm, specifically about 100 μm to about 2 mm, more specifically about 100 μm It may range from 1 mm.

Membrane filtering

In one embodiment, the supernatant separated through centrifugation described above is recovered and used as a detection target. However, it is difficult to apply the supernatant directly to subsequent processes (specifically, nucleic acid extraction through cell lysis), such as containing a large amount of liquid components compared to the amount of sample particles to be detected. Therefore, it is necessary to remove the liquid component and obtain sample particles in a concentrated form.

To this end, a pre-treatment step using a membrane filter can be performed. In FIG. 2, an exemplary structure of a membrane filter for separating the supernatant obtained by centrifugation into a retainer and a filtrate (filtrate) is shown.

Referring to FIG. 2, the membrane filter 100 largely includes an upper substrate A, a filter structure B, and a lower substrate C from the upper side.

For example, each of the upper substrate A, the frame of the filter structure B, and the lower substrate C may be, for example, a plastic material, specifically, a plastic material having light transmittance. Transparency does not substantially affect the filtering function, but it may be advantageous to use a plastic substrate having high transparency, if necessary, so that the operation of the membrane filter, the degree of filtering, and the like can be easily monitored. Exemplarily, the plastic material is polyester (specifically, polyethylene terephthalate (PET)), polyethylene (PE), polypropylene (PP), polyimide (PI), polystyrene (PS), polycarbonate (PC), poly Urethane, polyvinylidene fluoride, nylon, polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), cyclic olefin copolymer (COC), liquid crystal polymer (LCP), polyamide (PA), polyimide ( PI), poly (phenylene ether) (PPE), polyoxymethylene (POM), polyether ether ketone (PEEK), polyether sulfone (PES), polytetrafluoroethylene (PTFE), acrylic resin, etc. have. More specifically, it may be polyethylene terephthalate (PET), but is not limited thereto.

On the other hand, the injected supernatant is introduced into the filter structure (B), the first guide between the upper substrate (A) and the filter structure (B) selectively so that the supernatant can be smoothly introduced into the filter structure (B) The substrate 102 may be provided. At this time, the injection pressure of the supernatant is not particularly limited as long as an effective filtration process can be performed in the membrane filter, for example, about 10 kPa to 10 MPa, specifically about 50 kPa to 5 MPa, more specifically It may range from about 100 kPa to 1 MPa.

The first guide substrate 102 is provided with a channel in the form of a trench or a groove in the substrate to guide the supernatant injected from the upper substrate A to the cavity formed in the central region. The width of this channel may range, for example, from about 0.3 to 2 mm, specifically from about 0.5 to 1.5 mm, more specifically from about 0.8 to 1.2 mm, but this can be understood for illustrative purposes. In addition, the shape and size of the cavity formed in the first guide substrate 102 may be appropriately determined according to the shape and size of the mesh structure and the membrane sequentially arranged in the downward direction. As an example, the shape of the cavity may be circular, for example, and its size (diameter) may be, for example, about 10 to 30 mm, specifically about 15 to 25 mm, more specifically about 18 to 23 mm. Range.

The supernatant that has passed through the cavity of the first guide substrate 102 passes through the first intermediate support substrate 103 having the mesh structure of the filter structure B. The first intermediate support substrate 103 has a function to primarily support the membrane 105 disposed on the lower side, and may additionally filter out a food lump of an excessively large size that may be present in the supernatant. In this regard, the hole size of the mesh structure may be, for example, in the range of about 1 to 3 mm, specifically about 1.5 to 2.5 mm, and more specifically about 1.7 to 2.3 mm, but the present invention is not limited thereto. In addition, since the second intermediate supporting substrate 107 for fixing it is disposed on the lower side of the membrane 105, a mesh structure is formed similar to that of the first intermediate supporting substrate 103, so that it is filtered by the membrane 105 The filtrate (filtrate) is transferred to the lower substrate (C). At this time, the second guide substrate 108 corresponding to the first guide substrate 102 is also disposed between the second intermediate support substrate 107 and the lower substrate C, so that the filtrate (filtrate) is the second guide substrate 108 ) Through the cavity formed in the channel to be collected into the lower substrate (C).

According to the illustrated embodiment, the membrane 105 may be fixedly installed under the first intermediate support substrate 103. Illustratively, the third intermediate support substrate 104 is formed between the first intermediate support substrate 103 and the membrane 105 to form a cavity conforming to the membrane shape, so that the membrane 105 can be more easily fixed. This may be arranged, and similarly, between the membrane 105 and the second intermediate supporting substrate 107, a fourth intermediate supporting substrate 106 having a cavity conforming to the membrane shape may be disposed. Therefore, when the third and fourth intermediate supporting substrates 104 and 108 are combined, the membrane 105 can be accommodated by the cavity formed in each and can be easily fixed in a predetermined position.

On the other hand, the membrane 105 allows the concentration of food sample particles (or microbial-containing (attached) food sample particles) to be recovered as a retentate.

According to an exemplary embodiment, the membrane 105 may be made of a material having hydrophilicity, so as to typically pass an aqueous supernatant. Illustratively, the membrane is coated with cellulose nitrate, cellulose acetate (e.g., a mixture of cellulose triacetate and cellulose diacetate), mixed cellulose esters (e.g., including cellulose nitrate and cellulose acetate), coating It may be a material such as cellulose ester (for example, casting cellulose acetate on a non-woven polyester support), hydrophilic PTFE, nylon (for example, made by impregnating a polyester web with a nylon polymer), polycarbonate, etc. have.

The pore size (diameter) of the membrane 105 may be, for example, in the range of about 0.5 to 4 μm, specifically about 0.8 to 3.5 μm, and more specifically about 1 to 3 μm. In addition, the thickness of the membrane may range from, for example, about 100 μm to 1 mm, specifically about 100 to 500 μm, more specifically about 100 to 300 μm.

In addition, the moisture flow rate of the membrane may be, for example, in the range of about 50 to 400 mL / min / cm 2, specifically about 100 to 350 mL / min / cm 2, more specifically about 200 to 300 mL / min / cm 2 .

Meanwhile, in the illustrated embodiment, each of the members constituting the upper substrate (A), the membrane filter (B) and the lower substrate (C), and the membrane filter (B) is to be mutually bonded (or bonded) in an adhesive interposed manner. Can be. The adhesive is not particularly limited as long as it has sufficient adhesiveness to mutually bond each layer constituting the membrane filter. Illustratively, a rubber-based adhesive, an acrylic resin-based adhesive, a silicone-based adhesive, an optical-based adhesive, and a heat-sensitive adhesive may be used. In this case, the adhesive may be applied in the form of a single-sided or double-sided adhesive tape, more specifically a double-sided adhesive tape, and a pressure-sensitive adhesive tape, a thermally active adhesive tape, a chemically active adhesive tape, a photoactive adhesive tape, and the like can be exemplified.

Gene extraction through lysis

According to one embodiment, in the filtration process by the membrane, the step of extracting the gene by lysis (lysis) of microorganisms (specifically, pathogens such as bacteria) contained in the retained material may be performed.

According to an exemplary embodiment, the aforementioned microorganisms may be specifically food-borne pathogens, and more specifically, E. coli O157: H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , yellow It can be Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi , Cholera, Mycobacterium, Pertussis, Diphtheria, Typhoid, Pest, Hemolytic Streptococcus or Staphylococcus aureus, Specifically, E.coli O157: H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi, S. enteritidis, Y. enterocolitica, S. aureus, B. cereus , L. monocytognes , and the like.

In this regard, various methods known in the art can be applied for the dissolution of microorganisms, wherein the microorganisms collected under controlled temperature conditions are heated at about 70 to 95 ° C (specifically about 80 to 90 ° C). A method of alternately performing pulse on and pulse off using an ultrasonic sonicator, a method of using a lysis buffer, and the like are applicable.

According to a specific embodiment, a nucleic acid extraction method using a lysis buffer solution can be used. As an example of a buffer component that can be added to the lysis buffer, HEPES ((4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid), MOPS (3- (N-morpholino) -propane Sulfonic acid), N-tris (hydroxymethyl) methylglycine acid (Tricine), tris (hydroxymethyl) methylamine acid (Tris), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES) And acetate or phosphate-containing buffers (K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , and / or NaH ₂ PO ₄ ), or combinations thereof, but the present invention is not limited thereto. The concentration of the buffer component in the lysis buffer solution may be, for example, in the range of about 3 to 30 mM, specifically about 5 to 20 mM, and more specifically about 6 to 10 mM. Optionally, the lysis buffer may further contain proteases used in the art for nucleic acid extraction, such as protease K, trypsin, chymotrypsin, elastase, streptoglycine, and the like. According to an embodiment, for nucleic acid extraction, commercially available Helix one ^™ kit, G-spin ^™ nucleic acid extraction kit, easy-spin ^™ nucleic acid extraction kit, Pathogen-spin ^™ nucleic acid extraction kit, QIAamp  DNA Mini Kit or the like can be used.

In one embodiment, the dissolution temperature for the retained material may be selected in consideration of the type of enzyme used, etc., and may be adjusted using a heating block or the like, for example, about 50 to 98 ° C ( Typically about 70 to 95 ° C). The temperature range is described for illustrative purposes, but the present disclosure is not limited thereto.

Amplification of extracted genes

According to one embodiment, an amplification reaction is performed on the gene (nucleic acid) extracted through lysis. For the amplification reaction, amplification techniques known in the art, such as PCR, can be utilized. For example, the PCR method is described in Korean Patent No. 593687, etc., and the above-mentioned prior documents are included as reference materials of the present invention. As a PCR method, assembly-PCR, asymmetric PCR, digital PCR, endpoint PCR, inverse PCR, methylation-specific PCR, qualitative PCR, quantitative PCR, real-time (real-time) PCR, reverse transcription (RT) -PCR, and the like can be exemplified, and specifically, real-time PCR can be applied.

According to an exemplary embodiment, the step of amplifying a gene may be performed using a microfluidic gene amplification device, wherein the microfluidic gene amplification device may be a PCR device.

According to an exemplary embodiment, the microfluidic-based gene amplification apparatus may include a gene amplification unit, a heating member for controlling temperature required for a cycle process during the amplification process, and a control unit for controlling a gene amplification reaction.

In this regard, the gene amplification unit may include at least one microchannel type gene amplification chamber. According to a specific embodiment, the gene amplification unit covers the upper open trench or groove by allowing the upper sheet to join on the lower sheet having at least one trench or groove on which the upper side is open. Can form. At this time, the chamber for gene amplification receives a fluid containing the extracted gene and corresponds to a space where an amplification reaction occurs according to a temperature cycle.

On the other hand, the heating member is placed in contact with or adjacent (non-contact) the gene amplification unit to perform the function of supplying the heat energy required for the gene amplification reaction. As long as it can supply heat to the microfluidic-based amplification unit, It is not limited to a specific kind of heating means. As an example of such a heating means, a thermoelectric element such as a Peltier element, a heater using resistive heat, and the like can be exemplified, and its form may be a heating block. In the case of the non-contact method, a near-infrared radiation device, a far-infrared radiation device, a hot air fan, etc. may be exemplified.

In addition, the control unit for controlling the gene amplification reaction is responsible for controlling the entire process for gene amplification. For example, temperature control and amplification for gene amplification, such as driving the heating member or adjusting its temperature. It can perform functions such as controlling the processing time in individual steps of the reaction. In addition, the control unit may store and process data generated in the gene amplification process, and may perform a function of communicating with a separate terminal. In addition to these functions, it is also possible to control all members requiring electronic control related to the gene amplification reaction. The control unit may be implemented in the form of an electronic control circuit, a computer program stored in a specific recording medium for control, an application, or the like. Meanwhile, the control unit may further include a display unit and an operation unit to externally display the gene amplification reaction process, results, and control contents. An example of the display unit may be a liquid crystal display, but is not limited thereto, and a known display can be used. The operation unit may also apply a known input means such as a button type or a touch type. In this regard, conditions such as temperature and time of the sample fluid to be diagnosed can be set through the operation unit, and a set value of an amplification reaction is checked through a display unit, and changes in temperature, time, etc. as the amplification reaction progresses are confirmed. Can be.

 Figure 3 schematically shows a gene amplification unit 200 of a microfluidic-based gene amplification device for gene amplification in an exemplary embodiment.

Referring to the drawings, the gene amplification unit 200 may be in the form of a chip, and largely includes an upper sheet 201 and a lower sheet 202. At this time, an injection port (not shown) of the gene extract by lysis is formed at a portion of the upper sheet 201, and a channel is formed in the lower sheet 202 in a groove shape. According to the illustrated example, a guide channel 211 for guiding the injected extract to the vicinity of the chamber 213 is formed on the lower sheet 202, and a branch channel 212 for branching into the plurality of chambers 213 is formed. . Therefore, it is possible to perform an amplification reaction in a plurality of chambers by injecting the extract once.

According to an exemplary embodiment, the materials of each of the upper sheet 201 and the lower sheet 202 may be formed of the same or different materials, and may be used materials exemplified in connection with the membrane filter as a whole. However, a transparent PET or polycarbonate material may be advantageous to visually check the flow of the fluid.

In addition, the fine channels 211 and 212 may be formed in a straight line or a curved pattern (spiral, serpentine, zigzag, etc.), but are not limited to the straight line pattern shown in FIG. 3. The formation of such a microchannel may use laser cutting, cutting plotter processing, cutting printing, and the like, and details of each of these are known in the art, and detailed descriptions thereof will be omitted. The width and / or depth of the microchannel is not limited to a specific value, and may be mainly configured to have micrometer-level dimensions.

According to an exemplary embodiment, the size of the chamber 213 in which the amplification reaction takes place is such that the volume of the liquid component is, for example, about 10 to 30 μl, specifically about 15 to 25 μl, and more specifically about 18 to 22 μl It can be designed to be in scope, but this can be understood in an illustrative sense.

In addition, the gene amplification unit 200 may be formed with an inlet and an outlet (not shown) capable of discharging the amplification reaction product performed in the chamber 213 and extracting liquid is injected from the upper sheet 201, A fine valve (not shown) may be further provided to control the flow of fluid in the amplification unit, that is, inflow and outflow. In this regard, at least one valve hole (not shown) may be formed at a predetermined portion in the microchannel, and the microvalve may be inserted therein to prevent loss due to evaporation of fluid during amplification.

As described above, the gene extract to be amplified is injected into the amplification unit 200 through the injection port, reaches the chamber 213 along the microchannels 211 and 212, and can be discharged to the outside through the discharge port after the amplification reaction. Illustratively, the inlet and outlet may use mechanical drilling, laser drilling, chemical etching, etc., each of these details is known in the art.

Detection of microorganisms (pathogens or food poisoning bacteria)

As described above, amplification products of microorganisms in food samples can be diagnosed or detected by methods known in the art, for example, gel electrophoresis, ELGA (enzyme-linked gel assay), ECL (electrochmiluminescent) or the like can be used. However, the above method may alternatively use a detection method using fluorescence because there is an inconvenience in that a detection step must be separately performed for each sample in order to confirm the result of the amplification reaction.

According to an exemplary embodiment, the amplified product can be detected using a labeled probe, specifically a fluorescently labeled probe. Representative examples of such fluorescently labeled probes are umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tamara (TAMRA), Fluoroscein, Alexa, including dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots, phycoerythrin, fluorecein amidite (FAM), etc. Fluorescent materials, such as a floor system (alexa fluor) and a cyanine system (cyanine) containing Cy3, Cy5, Cy7, and indocyanine green, etc. are mentioned, and one or two or more of them can be used. However, the present invention is not limited to the fluorescent materials exemplified above. In this connection, the fluorescent material is excited by light of a specific wavelength, and then emits excess energy by emitting light of another wavelength. A fluorescent material such as FITC emits light of 550 nm wavelength.

In another embodiment, a detection method using fluorescence of a molecular beacon probe or a TaqMan probe can be used. Particularly, when the biochip according to the present invention is used, a fluorescence detection method using a molecular beacon or a TaqMan probe is particularly advantageous, because it is possible to perform concentration, amplification, and detection of a sample in a single biochip.

Molecular beacon probes are those in which the 5 'end of an oligonucleotide is coupled with a fluorescent dye and the 3' end with a quencher. The oligonucleotide strand is composed of a loop-type probe portion complementary to a target sequence, and self-complementary regions of 5 to 6 nucleotides at both ends thereof. In the absence of a target, the complementary portion of the probe is in the form of an adjacent hairpin. The phosphor and the matting agent are adjacent to each other to show a strong quenching effect. On the other hand, when it reacts with the target, the loop-like arrangement is opened and becomes a straight line, so that the distance between the phosphor and the matting agent is increased, and fluorescence is observed.

In the case of the TaqMan probe, an oligonucleotide modified with a 5 'end with a phosphor (FAM, etc.) and a 3' end with a quencher (TAMRA, etc.) is added to the amplification solution.The TaqMan probe is specific to the template nucleic acid in the annealing step. Although it hybridizes, fluorescence is suppressed by the quencher on the probe. As the TaqMan probe hybridized to the template is decomposed by the activity of the 5 '→ 3' nucleic acid endonase (exonuclease) of the Taq DNA polymerase during the extension reaction, the fluorescent dye is released from the probe and the inhibition by the quencher is released. It shows fluorescence. However, it may be more preferable to use molecular beacons in terms of detection specificity.

The above-described probe is used in addition to the amplification solution. When added prior to the amplification, the primer may form a competitive relationship with the probe in binding to the nucleic acid. That is, non-specific binding may be increased by hybridization of the probe and the target nucleic acid. Therefore, when an excessive amount is used, it can act as an inhibitor of the amplification reaction. On the other hand, when the amount of the probe is less than a certain level, it is difficult to expect a desired detection effect due to insufficient fluorescence characteristics compared to the amplified nucleic acid. After all, it is preferable to experimentally properly adjust the amount of the probe and other reaction conditions in consideration of the amplification method. In order to eliminate the possibility of this problem, for example, by increasing the amplification reaction time, it is possible to increase the maximum limit amount of the probe while suppressing the effect of the probe addition on the amplification reaction.

According to another exemplary embodiment, an insert dye may be added to the amplification mixture prior to or during the amplification step. At this time, when amplifying DNA as a nucleic acid, the insertion dye is inserted between double stranded DNA (dsDNA), and the amplification degree can be confirmed by measuring the intensity according to fluorescence detection. In this regard, the insert dye is a dye that can be inserted into a double strand in the amplification process, SYBR Green, PicoGreen, Hoechst series, BOBO, TOTO, YOYO, JOJO, POPO, LOLO, PO-PRO, BO-PRO, YO -PRO, TO-PRO, JO-PRO, LO-PRO, SYTO series, etc., and these may be used alone or in combination. Typically, SYBR Green, TOTO-3 from the TOTO series, POPO-3 from the POPO series, BOBO-3 from the BOBO series, and the like can be used. The present invention is not necessarily limited to the above-described types, and various types may be used as long as it can be inserted into a double strand during the amplification reaction and detect fluorescence characteristics derived therefrom. In addition, the amount of the insert dye to be added can be appropriately adjusted in consideration of the properties of the insert dye, for example, may be in the range of about 0.1 to 20 μM weight% (specifically, about 1 to 5 μM). However, the present disclosure is not limited to the above numerical range.

As described above, the fluorescence derived from the amplification product is, for example, a confocal microscope, a fluorescence microscope, a fluorometer, a fluorescence scanner, a flow cytometer. ) Can be confirmed through detection means (or devices) known in the art, and the presence and concentration of the target nucleic acid can be analyzed in consideration of the presence and intensity of fluorescence. According to an exemplary embodiment, for fluorescence detection, for example, a laser-induced confocal fluorescence microscope can be used. In addition, any equipment capable of confirming the presence or absence of fluorescence and the change in fluorescence intensity according to the presence of a nucleic acid (target nucleic acid) can be used without particular limitation, for example, QuantaMaster PTI spectrofluorimeter (PTI), SPEX Fluorolog 3 (ISA), etc. can do.

The detection limit (LOD) of the diagnostic system according to the exemplary embodiment may be, for example, in the range of about 10 to 10 ² CFU / ml, but is not limited thereto.

Hereinafter, preferred embodiments are provided to help understanding of the present invention, but the following examples are provided only to more easily understand the present invention, and the present invention is not limited thereto.

Example 1

In this embodiment, the membrane filter and the microfluidic device shown in FIGS. 2 and 3 are used, and the structure and material thereof are as follows:

-Membrane filter: It was manufactured and used according to its own design using a commercially available polycarbonate polymer.

-Membrane: Hydrophilic membrane with pore size of 3 μm

-Double-sided tape: After processing to a certain shape using a custom-made double-sided adhesive film, it was applied to adhesion when manufacturing membrane filters and microfluidic devices.

-Polyethylene terephthalate (PET) film: A high transparency film was used.

The dimensions of the membrane filter shown in FIG. 2 are shown in Table 1 below.

Composition absence size Upper and lower substrates (A, C) 40 mm × 40 mm First and second guide substrates 102 and 108 Channel 1 mm, cavity diameter: 18 mm First and second intermediate support substrates 103 and 107 Mesh hole size: 2 mm Membrane (105) Pore size: 3㎛ Third and fourth intermediate support substrates 104, 106 Cavity diameter: 18 mm

In addition, the total size in the amplification part of the microfluidic device shown in FIG. 3 was 40 mm × 40 mm, and the volume of the nucleic acid extract contained in each of the four amplification chambers was 20 μl.

The samples used in the examples were prepared as follows.

-Samples containing bacteria

E.coli O157: H7 ((ATCC 43895): American Type Culture Collection (Washington DC, USA)) was selected and the pathogen was placed in a tube and stirred at 37 ° C. at 250 rpm with 15 mL of Luria-Bertani (LB) medium. While incubating for 24 hours. At this time, when the optical density (OD) value by the UV absorbance measurement method (600 nm) was 1, the culture was terminated to recover the culture, diluted to a desired concentration, and injected into a commercially available instant food product. . At this time, triangular gimbap sold in convenience stores was purchased and used as instant food (brand name: Spam & Tuna Mayo 110g (Ministop) Spam & Kimchi Stir-Fry 100g (Ministop), Tuna Mayonnaise 110g (Seven Eleven), Jeonju Bibimbap 105g ( Seven-Eleven) and stir-fried 145g (mini-stop). The purchased instant food was placed in a zipper bag and mixed evenly by hand.

 After adding 25 g of each triangular gimbal and 20 ml of 1 ㅧ PBS to a 50 ml tube and shaking it for about 30 times, it was put in a mesh (sieve) and pressed to reduce the size of the contents.

Thereafter, the obtained sample was transferred to a new tube, and E. coli having a copy number of 10 ⁸ was serially diluted (serial dilution). Specifically, 90 μl of PBS was added to 10 1.5 ml tubes, and 10 μl of E. coli 10 ⁸ was added to 2 tubes, followed by vortexing so that the E. coli concentration was 10 ⁷ . Then, 10 μl was taken out of the pipette from E. coli 10 ⁷ and put into two other tubes, respectively, and then vortexed to adjust the E. coli concentration to 10 ⁶ . By repeating the above procedure, samples having an E. coli concentration of 10 ⁵ to 10 ² were prepared. That is, through the above-described procedure, samples of up to 10 ⁷ to 10 ⁰ for each of five instant foods were prepared.

-Centrifugation

E. coli was added to 10 μl of each copy number (10 ⁶ to 10 ² ) in a tube containing each sample, and centrifugation was performed under the conditions shown in Table 2 below.

division Condition Rotation speed 2000 rpm Centrifugation time 30 seconds Temperature 4 ℃

-Membrane filtering

Following centrifugation, the supernatant was removed from the supernatant and the contents separated into pellets, injected into a membrane filter, and the retained matter remaining on the membrane was recovered. At this time, the supernatant was injected in a manner using a micropipette.

-Nucleic acid extraction

The retention of the membrane filtering obtained as described above was placed in a 1.5 ml tube and a Helix one ^™ kit having the composition of Table 3 below was added as a cell lysis reagent.

division Condition DNA-free water 69 μl 10X buffer solution 10 μl Helix one 1 μl

 Thereafter, after vortexing, the temperature was adjusted according to a heating protocol as shown in Table 4 below using a heating block.

division Temperature time Step 1 75 ℃ 5 minutes Stage 2 95 ℃ 5 minutes

Nucleic acids were extracted from microorganisms ( E. Coli ) according to the procedure described above. At this time, the concentration of bacteria in the sample was changed from 10 ⁷ cell / mL to 10 ⁰ cell / mL, and deionized water was used as a negative control. For each of these, i-StarMAX ^™ having the composition shown in Table 5 below was used as an amplification reagent, and amplification reaction was performed using a microfluidic PCR device.

Furtherance Add vol. (Μl) Deionized water 12.5 10X reaction buffer 2 Forward primer One Reverse primer One dNTP 2 Extracted viral lysate One polymerase 0.5

At this time, the gene amplification conditions were 95 cycles of 10 seconds (dissociation step), 58 ° C 5 seconds (combination step), and 72 ° C 10 seconds (extension step) with a total of 35 cycles (cycle). It was repeated, and then it was a method of performing an extension step at 72 ° C. for about 2 minutes.

In order to prepare an agarose gel (2%) for electrophoresis analysis of the amplified gene, 100 mL of 1X TAE buffer and 2 g of Agarose LE (intron, Cat No. 32034) were mixed and then mixed in a microwave oven. After melting, a fluorescent dye (red safe) was added to the mixture, and then poured into a gel plate to solidify.

In the electrophoresis gel experiment, 6 µl of the amplified product sample by PCR and 1 µl of the 6X loading buffer were mixed, and then 5 µl was loaded onto the 2% agarose gel prepared previously, and the gel was placed on the electrophoresis apparatus for 20 minutes. After dropping, it was measured by UV. At this time, the standard gene was extracted and the gene amplification products were compared under the same conditions, and the results are shown in FIG. 4.

According to the figure, in the image obtained through the gel electrophoresis experiment, the left image is the result of extracting DNA, while the right image is the result obtained from the gene amplification product according to the embodiment. That is, in the case of the standard sample (left), it was possible to detect from 10 ⁷ cell / mL to 10 ⁰ cell / mL, and in the example, it was confirmed that it was detectable from 10 ⁷ cell / mL to 10 ¹ cell / mL.

Example 2

A triangular gimbap (tuna mayonnaise) sample as an instant food was injected into the membrane filter by centrifugation as in Example 1, and the cell lysis buffer solution was added to the retained product to recover bacteria in the sample. Quantitative polymerization element chain reaction (Real time PCR) detection test was performed according to the concentration of ( E. coli ). At this time, the bacterial concentration in the sample was changed from 10 ⁷ cell / mL to 10 ⁰ cell / mL, and deionized water was used as a negative control.

Specifically, after injecting the gene extract into each tube, a detection experiment was conducted through quantitative analysis by concentration using real-time PCR. At this time, the conditions of real-time PCR are shown in Table 6 below.

Furtherance Add vol. (Μl) Deionized water 7 Forward primer One Reverse primer One RbTAG premix 10 Extracted viral lysate One

As a result of the detection test, Ct values are shown in Table 7 below, and fluorescence characteristics are shown in FIG. 5 together with a standard curve. In addition, the results obtained by changing the rotational speed in the centrifugation process to obtain the supernatant in the above table and drawings to 500 rpm and 1000 rpm, respectively.

10 ⁷ 10 ⁶ 10 ⁵ 10 ⁴ 10 ³ 10 ² 10 ¹ 10 ⁰ Standard curve 14.85 14.61 20.33 22.3 26.65 29.53 33.56 36.97 2000rpm 20.93 24.09 27.69 30.78 33.49 2000rpm 19.85 22.35 26.57 30.23 32.15 1000rpm 23.19 25.42 29.56 32.33 32.18 1000rpm 21.26 25.13 28.36 30.28 31.96 500rpm 21.01 25.08 28.43 31.71 32.53 500rpm 21.23 25.18 28.73 31.41 32.51

Referring to the above table and drawings, the illustrated graph shows the results for real samples based on real time PCR results based on standard samples. Specifically, in the embodiment, as the amount of the sample actually injected increases, the gene amplification progresses rapidly and shows a relatively low Ct value. Ct value. As such, quantitative and / or qualitative analysis is possible according to the concentration of DNA, and based on this, a range of results that can be detected in real time PCR can be expressed.

According to the above table and drawings, the fluorescence characteristics of the real time PCR according to the examples are almost in line with the standard curves, indicating that the detection results according to the examples secure reliability. Therefore, when the detection platform according to the present embodiment is applied, it is possible to effectively diagnose even a low concentration of bacteria-containing samples.

Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (19)

a) preparing a sample-containing buffer solution by injecting a water-based salt-containing buffer solution into a textured texture carbohydrate-containing food sample, and then reducing the size of the sample in the sample-containing buffer solution to 10 mesh or less;
b) The sample-containing buffer solution is centrifuged under a rotational speed condition of 100 to 5000 rpm to separate into a water-based supernatant containing a food sample in the form of a particle having a size of 1 mm at 100 μm and a pellet on the lower side. Letting;
c) pre-treating the supernatant through a membrane filter to form a concentrate of the particulate food sample as a retentate of the membrane filter;
d) lysing the retainer of the membrane filter to extract a gene from the retainer;
e) amplifying the extracted gene; And
f) quantitatively, qualitatively or quantitatively and qualitatively detecting microorganisms from the gene amplification products produced as a result of step e);
Included, The membrane filter,
An upper substrate on which an injection hole of the supernatant is formed;
A filter structure positioned below the upper substrate and having a hydrophilic membrane interposed between the first and second intermediate support substrates having a hole-sized mesh structure of 1 to 3 mm; And
A lower substrate positioned under the filter structure and having a discharge channel for discharging the filtrate filtered by the filter structure;
Method for detecting a pathogen from a food containing a. delete The method of claim 1, wherein the step of reducing the size of the sample is performed using sieving, crushing, grinding using a blender or mixer, or a combination thereof. The method of claim 1, wherein the food sample is an instant food sample, characterized in that at least one is selected from the group consisting of rice and bakeries. According to claim 1, Between the upper substrate and the filter structure is interposed a first guide substrate having a channel and a cavity for guiding the supernatant injected from the upper substrate to the filter structure,
And a second guide substrate having a channel and a cavity for guiding the filtrate to the lower substrate between the filter structure and the lower substrate. The method of claim 5, wherein the first intermediate support substrate and the membrane, and between the membrane and the second intermediate support substrate, a cavity having a shape conforming to a membrane is formed to receive and fix the membrane in combination. And a fourth intermediate support substrate interposed. The method of claim 1, wherein the pore size (diameter) of the membrane is 0.5 to 4 μm, and the thickness of the membrane is 100 μm to 1 mm. The method according to claim 1, wherein the membrane is made of cellulose nitrate, cellulose acetate, mixed cellulose ester, coated cellulose ester, hydrophilic PTFE, nylon or polycarbonate. The method of claim 1, wherein step e) is performed using a microfluidic gene amplification device. The method of claim 9, wherein the microfluidic gene amplification device,
(i) a gene amplification unit having at least one microchannel type gene amplification chamber;
(ii) a heating member disposed in contact with or adjacent to the gene amplification unit to supply heat energy necessary for a gene amplification reaction; And
(iii) a control unit for controlling the gene amplification reaction;
Method comprising a. 11. The method of claim 10, The gene amplification unit At least one trench or groove (groove) is formed at the top of the lower sheet is formed; And bonded to the lower sheet to cover at least one trench or groove in which the upper side is opened to form a chamber for gene amplification,
The method for gene amplification is characterized in that it provides a space for receiving a gene containing the extracted gene and a gene amplification reaction space. The method of claim 10, wherein the gene amplification unit further comprises a micro-valve for controlling the inflow and outflow of fluid introduced into the micro-channel. The method of claim 1, wherein the pathogen is E. coli O157: H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi Method characterized in that it is cholera, isolate, pertussis, diphtheria, typhoid, pest, hemolytic streptococci or Staphylococcus aureus. The method of claim 1, wherein the sample-containing buffer solution is prepared by injecting 10 to 150 parts by weight of the buffer solution with respect to 100 parts by weight of the food sample. The method of claim 1, wherein in step b), the centrifugation temperature is 2 to 15 ° C, and the centrifugation time ranges from 20 to 50 seconds. The method of claim 1, wherein step d) is performed by adding a lysis buffer solution to the retained material. 17. The method of claim 16, wherein step d) is performed at 50 to 98 ° C. The method of claim 1, wherein step e) is performed by real-time PCR. a ') A sample-containing buffer solution is prepared by injecting a water-based salt-containing buffer solution into a texture textured carbohydrate-containing ready-to-eat food sample selected from at least one of rice and bakery, and then the sample-containing buffer solution Reducing my sample size to 10 mesh or less;
b ') The sample-containing buffer solution is centrifuged under a rotational speed condition of 500 to 2000 rpm to separate into a water-based supernatant containing a food sample in the form of particles having a size of 100 µm to 1 mm and a pellet on the lower side. Letting;
c ') The supernatant is pretreated through a membrane filter equipped with a membrane having a pore size of 0.5 to 4 μm and a thickness of 100 to 300 μm to retain the concentrate of the food sample in the form of a membrane filter ( retentate);
d ') extracting a gene from the retained material by adding a lysis buffer solution to the retained material of the membrane filter to dissolve;
e ') performing PCR amplification on the extracted gene using a microfluidic gene amplification device; And
f ') quantitatively, qualitatively, or quantitatively and qualitatively detecting food poisoning bacteria from the gene amplification product produced as a result of step e');
Included, The membrane filter,
An upper substrate on which an injection hole of the supernatant is formed;
A filter structure positioned below the upper substrate and having a hydrophilic membrane interposed between the first and second intermediate supporting substrates having a hole-sized mesh structure of 1 to 3 mm; And
A lower substrate positioned under the filter structure and having a discharge channel for discharging the filtrate filtered by the filter structure;
Method for detecting food poisoning bacteria from ready-to-eat food containing.

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