KR101910172B1 - Method for Detection of Food Poisoning Bacteria By Using Gene Amplification and Kit for Use in The Same Method - Google Patents

Abstract

The present invention relates to a method for detecting food poisoning-causing bacteria using a gene amplification method and a kit used in the method. The method of the present invention extracts a nucleic acid from a sample and concentrates the extracted small amount of nucleic acid through WGA (Whole Genome Amplification) reaction, thereby accurately and rapidly detecting a trace amount of food poisoning- It has an effect that can be detected.

Description

[Method for Detection of Food Poisoning Bacteria By Using Gene Amplification and Kit for Use in The Same Method}

The present invention relates to a method for detecting food poisoning-causing bacteria using a gene amplification method and a kit used in the method. More specifically, the present invention relates to a method for rapidly and accurately detecting a trace amount of food poisoning-causing bacteria contaminated with fish through a molecular concentration method and a gene amplification method, and a kit used in the method.

Aquatic organisms, which are often eaten raw, such as sashimi, often cause diseases such as food poisoning by microbes (bacteria, viruses, etc.) contaminated in trace amounts. According to the current Food Code, in the case of raw fish, in order to detect microorganisms such as food poisoning bacteria, microorganisms collected from fish tissues are cultured in a selective medium to isolate bacteria that are supposed to be food poisoning bacteria, and then biochemically or immunologically. It is to be confirmed by the academic method.

Although general immunological methods can detect bacteria with high accuracy, a large amount of sample is required to detect food poisoning bacteria that are contaminated in trace amounts. In addition, in order to produce antibodies specific to each microorganism, purification of the bacteria, Antibody production or peptide production is essential, and thus high antibody production costs are required. Here, due to the nature of the protein, it is difficult to store and utilize it, and because there are many cases where only one type or limited detection is possible at a time, there are many restrictions on use, and a long time is consumed in the cultivation and experimentation of bacteria.

In order to improve these shortcomings, research and development of various bacterial detection kits using a polymerase chain reaction (PCR) method have begun. Detection kits using the polymerase chain reaction method are increasingly in demand in various fields due to their high accuracy, simplicity, and rapidity. In particular, the Real-Time Polymerase Chain Reaction (PCR) method, which has been widely used in recent years, is a method of observing the increase of nucleic acids in real time every polymerase chain reaction cycle. It is a method of interpretation by detection and quantification. This method has excellent accuracy and sensitivity, has high reproducibility, can be automated, can quantify results, is quick and simple, and is harmful to humans such as contamination by nucleic acid dyeing materials such as EtBr (Ethidium Bromide) and UV irradiation. It has the advantage of being less exposed to the substance, and has the advantage of being able to automatically check the presence or absence of amplification of a specific gene. In addition, through the real-time polymerase chain reaction method, it is possible to confirm quantitative results with high specificity rather than qualitative results such as end-point polymerase chain reaction or antigen/antibody reaction. In addition, since a probe labeled with a fluorescent labeling factor is used, the result can be confirmed with a sample of less than the amount of the sample used for the DNA chip or antigen/antibody reaction.

However, in order to measure through the polymerase chain reaction method in micro-contaminated microbes, the amount of contaminated micro-organisms is large, or it takes time to increase the number of strains that can be detected. Due to the nature of aquatic organisms, the amount of samples is small and spending time through cultivation is not suitable for the purpose of detecting contaminants such as food poisoning bacteria. There is a need for a system that can detect and diagnose this as a whole.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Korean Patent Registration No. 10-1299627 Korean Patent Registration No. 10-0919261 Korean Patent Registration No. 10-1100932

The present inventors have made research efforts to develop a method and system for detecting food poisoning-causing bacteria present in trace amounts in foods such as fish based on a genetic diagnosis method. As a result, by developing a method that can quickly and accurately detect the nucleic acids of food poisoning-causing bacteria that are present in foods such as fish in trace amounts, and after molecular concentration, real-time polymerase chain reaction can be used. The invention was completed.

Accordingly, an object of the present invention is to provide a method for detecting food poisoning-causing bacteria using a gene amplification method.

Another object of the present invention is to provide a kit for real-time polymerase chain reaction for detecting food poisoning-causing bacteria.

Objects and advantages of the present invention will become more apparent from the following detailed description, claims, and drawings.

According to one aspect of the present invention, the present invention provides a method for detecting food poisoning-causing bacteria using a gene amplification method comprising the following steps:

(a) dissolving the bacteria causing food poisoning to be detected, and then binding the nucleic acids of the bacteria to be detected to magnetic beads;

(b) washing the magnetic beads to which the nucleic acid is bound to remove contaminants;

(c) eluting the nucleic acid from the magnetic beads to which the nucleic acid is bound;

(d) molecularly concentrating the eluted nucleic acid; And

(e) performing real-time gene amplification on the molecularly-enriched nucleic acid.

Hereinafter, the method of the present invention will be described in detail by dividing it into each step.

Step (a): After dissolving the bacteria causing food poisoning to be detected, the nucleic acid of the bacteria to be detected is bound to magnetic beads.

In the method of the present invention, first, the detection target food poisoning-causing bacteria is dissolved to induce the genomic nucleic acid to escape from the cell. In the present invention, the food poisoning-causing bacteria include, for example, Vibrio spp., Staphylococcus aureus, Salmonella spp., and Listeria monocytogenes. In the present invention, the sample to be detected is a sample estimated to have been infected with the food poisoning-causing bacteria, and may be, for example, blood or fish, but is not limited thereto.

The lysis buffer used to dissolve the bacteria to be detected in the present invention includes a Chaotropic reagent, a surfactant, EDTA, and Tris. The Chaotropic reagent includes, for example, guanidine thiocyanate or guanidine hydrochloride. Triton X-100, zwitterionic detergent, or CHAPS may be used as the surfactant, but is not limited thereto.

Magnetic beads capable of binding to nucleic acids are added to the solution in which the cells are dissolved, thereby inducing binding of the nucleic acids to the magnetic beads. The nucleic acid is reacted with the magnetic beads to bind the nucleic acid to the magnetic beads, and the magnetic beads to which the nucleic acids are bound are separated from the solution. Separation of the magnetic beads may be performed by applying a magnetic field to the magnetic beads to fix the magnetic beads and then removing the solution from the magnetic beads.

Step (b): removing contaminants by washing the magnetic beads to which the nucleic acid is bound

After separating the magnetic beads to which the nucleic acid is bound from the solution, the contaminants bound to the magnetic beads are washed. Washing of the contaminants is performed using a washing buffer. The wash buffer contains one or more components of ethanol, isopropanol and salt.

Step (c): eluting the nucleic acid from the magnetic beads to which the nucleic acid is bound

As described above, the magnetic beads to which the nucleic acids are bound are washed with a washing buffer, and then the nucleic acids are separated from the magnetic beads and eluted. The nucleic acid is eluted using an elution buffer.

Step (d): molecularly concentrating the eluted nucleic acid

Molecular concentration of the eluted nucleic acid is carried out through the WGA (Whole Genome Amplification) method. The WGA used for molecular enrichment in the present invention includes both a PCR-based WGA method and a Multiple Displacement Amplification (MDA) method, and more preferably, molecular enrichment through an MDA-based method is used. In the WGA method for molecular concentration of nucleic acids of the present invention, WGA may be performed by adding a substance exhibiting a macromolecular crowding effect. The material exhibiting the macromolecular crowding effect may be, for example, polyethylene glycol (PEG), but is not limited thereto.

Step (e): performing real-time gene amplification on the molecularly-enriched nucleic acid

The molecularly-enriched nucleic acid is subjected to real-time gene amplification using this as a template to diagnose the presence or absence of a target bacterium. According to an embodiment of the present invention, in the present invention, Vibrio spp., Staphylococcus aureus, Salmonella spp., Listeria monocytogenes are detected in the present invention. In this case, a real-time polymerase chain reaction of the TaqMan method using the primers and probes disclosed in SEQ ID NOs: 1-36 is carried out.

According to another aspect of the present invention, the present invention provides a food poisoning-causing bacterium, Vibrio spp. bacteria, Staphylococcus aureus (Staphylococcus aureus), Salmonella genus ( Salmonella spp.) provides a real-time polymerase chain reaction kit for detecting bacteria or Listeria monocytogenes:

A first primer consisting of SEQ ID NOs: 1, 2 and 3-a probe set;

A second primer consisting of SEQ ID NOs: 4, 5 and 6-a probe set;

A third primer consisting of SEQ ID NOs: 7, 8 and 9-probe set;

A fourth primer consisting of SEQ ID NOs: 10, 11 and 12-probe set;

A fifth primer consisting of SEQ ID NOs: 13, 14 and 15-probe set;

A sixth primer consisting of SEQ ID NOs: 16, 17 and 18-probe set;

A seventh primer consisting of SEQ ID NOs: 19, 20 and 21-probe set;

An eighth primer consisting of SEQ ID NOs: 22, 23 and 24-probe set;

A ninth primer consisting of SEQ ID NOs: 25, 26 and 27-a probe set;

A tenth primer consisting of SEQ ID NOs: 28, 29 and 30-probe set;

An eleventh primer consisting of SEQ ID NOs: 31, 32 and 33-a probe set; And

The twelfth primer-probe set consisting of SEQ ID NOs: 34, 35 and 36.

As used herein, the term "primer" is complementary to the 5'-end sequence or the 3'-end sequence of the target nucleic acid site to be amplified during the nucleic acid amplification reaction, and in a suitable buffer at a suitable temperature ( That is, it means a single-stranded oligonucleotide capable of serving as an initiation point of a polymerase reaction of a template-indicating nucleic acid under four different nucleoside triphosphates and polymerase).

The term "probe" as used herein is a single-stranded nucleic acid molecule, and includes a sequence complementary to a target nucleotide sequence. According to an embodiment of the present invention, the probe of the present invention can be modified within a range in which the advantages of the probe of the present invention, that is, hybridization specificity, are not impaired. For example, a reporter fluorescent substance or a fluorescent inhibitor (quencher) may be tagged to the end of the probe oligonucleotide.

In the real-time polymerase chain reaction of the present invention, the probe uses a probe capable of complementarily binding to a partial sequence inside a nucleotide sequence amplified by a primer pair.

According to a preferred embodiment of the present invention, a reporter-fluorescent material or a fluorescence inhibitor (quencher) is tagged at the 5'-end and 3'-end of the probe used in the real-time polymerase chain reaction (PCR) of the present invention. I can.

In the method of the present invention, various DNA polymerases can be used in the amplification reaction, and preferably, the DNA polymerase is a thermostable DNA polymerase that can be obtained from various bacterial species. These include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).

On the other hand, when carrying out the gene amplification reaction, it is preferable to provide an excess amount of components necessary for the reaction in the reaction vessel. The excessive amount of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component. Cofactors such as Mg2+, dATP, dCTP, dGTP and dTTP are required to be provided to the reaction mixture to the extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

According to another preferred embodiment of the present invention, the kit further comprises a buffer solution, a DNA polymerase, a DNA polymerase cofactor, and dNTPs.

The kit of the present invention optionally includes reagents necessary for nucleic acid amplification, such as buffers, DNA polymerases (e.g., Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)). Thermostable DNA polymerase obtained from), DNA polymerase cofactors, and dNTPs.

The kit of the present invention may be manufactured in a number of separate packaging or compartments including the reagent components described above.

The present invention relates to a method for detecting food poisoning-causing bacteria using a gene amplification method and a kit used therefor. The method of the present invention extracts a nucleic acid from a sample and molecularly concentrates the extracted trace amount of nucleic acid through a WGA (Whole Genome Amplification) reaction, thereby accurately and quickly removing trace amounts of food poisoning-causing bacteria contaminated with fish used as raw fish It has a detectable effect.

1A is a diagram showing a schematic diagram of a nucleic acid extraction method used in the present invention.
1B shows the results of extracting nucleic acids using magnetic particles for each manufacturer.
2 is a diagram comparing the difference in the amount of nucleic acid extraction according to the amount of lysis buffer through an agarose gel electrophoresis experiment.
Figure 3 is a table comparing the nucleic acid extraction degree of the control group and the experimental group through a nanodrop spectrophotometer measurement value. The control group is a conventional column type nucleic acid extraction method, and the experimental group is a result of using a magnetic bead type nucleic acid extraction method.
FIG. 4 is a diagram comparing the nucleic acid extraction levels of the control group and the experimental group measured in FIG. 3 through an agarose gel electrophoresis experiment. Unlike the absorbance value, it was found that the amount of nucleic acid in the experimental group was large, and it was confirmed that substances that affect the absorbance value in the nucleic acid extraction solution were relatively less contaminated in the experimental group than in the control group.
FIG. 5 is a diagram showing the results of an agarose gel electrophoresis experiment in which an elution buffer was added to elute the nucleic acid bound to magnetic nano-particles and the time required for elution was confirmed.
6 is a table showing measured values of a nano-drop spectrophotometer in which an elution buffer was added to elute a nucleic acid bound to a magnetic nano-particle, and the time required for elution was checked.
FIG. 7 shows an agarose gel electrophoresis experiment of 25 ng each based on the measured value of a nano-drop spectrophotometer after extracting the nucleic acid under the same conditions as other products in order to compare the quality of the extracted nucleic acid. This is the picture compared.
8 is a diagram for comparison using a multiplex polymerase chain reaction experiment method in order to compare the quality of the nucleic acid used in FIG. 7.
9 is a diagram showing the results of an experiment using a real-time gene amplification experiment technique for the quantification value of the nucleic acid extracted for each company using human genomic DNA, in which the amount of nucleic acid was quantified, as a control.
10 is a schematic diagram illustrating a Multiple Displacement Amplification (MDA) technique used for molecular concentration.
11 is a result of quantifying the amount of nucleic acid extracted from a trace amount of microorganisms through Picogreen quantitative analysis.
12 is a result of the WGA test using a Crowding reagent. This is the result of electrophoresis on an agarose gel after reacting for 30 minutes.
13 is a result of measuring the effect on the WGA response according to the presence or absence of PEG through a real-time gene amplification method. This is the result of sequentially diluting the extracted nucleic acid and confirming the amount of nucleic acid increase with or without PEG.
14 is a diagram showing the results of testing nucleic acids extracted from harmful bacteria in a real-time gene amplification diagnostic kit. This is the result of real-time gene amplification by diluting the extracted nucleic acid through sequential dilution in an amount of 1/10 and using it as a template.
15 is a result of testing the nucleic acid extracted from harmful bacteria in the corresponding diagnostic kit SET A, and shows the results showing that only Vibrio and Salmonella are detected.
16 is a result of testing the nucleic acid extracted from harmful bacteria in the corresponding diagnostic kit SET B, and shows the results showing that only Staphylococcus and Listeria are detected.
17 is a result showing that a trace amount of nucleic acid can be measured within a short time through nucleic acid amplified through molecular concentration. As for the nucleic acid used for molecular concentration, 1/200 of the nucleic acid used in FIGS. 15 and 16 was used, and the results shown are figures showing similar results based on the Ct value.
18A and 18B are a comparison of the efficiency of molecular concentration. As a result of molecular concentration using only the extracted nucleic acid for DNA, and for normal WGA using a previously sold WGA kit, enhanced WGA is the result of molecular concentration using PEG. Is one of them. It can be seen that the Ct value of the enhanced WGA using PEG is higher than that of the case of using only DNA, and that the Ct value of the enhanced WGA using PEG is more advanced than that of the general WGA result, and it is the result showing that the molecular concentration is possible effectively.
19 is a result showing the reduction of detection time indicated by molecular concentration and a molecular diagnostic kit developed for the detection of food poisoning bacteria. , eWGA is a result showing the enhanced WGA. The result shows that the amount of nucleic acid used in the reaction is the same, and the amount of nucleic acid entering the real-time PCR is constant and detection is possible within a short time by molecular concentration.
FIG. 20 is a result showing that the UDG system was applied to the reaction reagent, and the previous reaction product (U+T PCR product) was removed from the reaction solution so as not to affect the reaction and thus no longer reacted.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Selection step of magnetic beads to be used for extraction of nucleic acids

First, a magnetic bead for each manufacturer to be tested was selected, and an experiment was conducted to compare the performance of each product shown in Table 1 below. The detection target bacteria were dissolved and the degree to which each product was bound was measured. Magnetic beads to be applied to the nucleic acid extraction reagent were selected based on the results measured by each manufacturer.

The solution used in the experiment was the reagent contained in the SolGent™ Genomic DNA Prep Kit (Solgent. co. Ltd, SGD61-S120, Korea), and 200 µl of whole blood was put into an Ependorf 1.5ml tube and SGB Lysis Buffer (Solgent, Korea) After adding 1 ml, the inverting mix was performed for 30 seconds, and then allowed to stand on ice for 10 minutes. After standing, vortex mixing was performed for 1 minute, and centrifugation was performed for 5 minutes in a centrifuge (Eppendorf, 5415D, Germany) at a speed of 12,000 rpm (revolutions per minute). After removing the supernatant, 800 µl of SGD2 (Solgent co. Ltd, Korea) solution was added to each solution, and then the pellet was suspended through vortex mixing. After adding 100 µl of magnetic particles for each manufacturer to 1 ml of the suspended solution, mixing through vortex mixing for 10 seconds, and then standing for 3 minutes on a magnetic stand, The solutions were separated from each other. After removing the separated solution as much as possible, 70% ethanol (100% ethanol 70ml + D.D.W 30ml) was added at 500µl per tube, followed by vortex mixing and suspended. The suspended tube was allowed to stand on a magnetic stand for 3 minutes to separate the solution and magnetic particles from each other. The separated solution was removed as much as possible, and the process of washing by adding 70% ethanol was repeated once more (a total of two times) to remove impurities attached to the magnetic beads. After the ethanol-removed tube was allowed to stand at room temperature for 5 minutes to remove the remaining ethanol, 50 µl of TE (Tris-EDTA, pH 8.0) buffer was added each to perform vortex mixing for 5 seconds. Thereafter, after standing on a magnetic stand for 3 minutes, the separated supernatant was transferred to a new 1.5 ml Eppendorf tube, and the purity and amount of DNA were measured. 1B shows the results of extracting nucleic acids using magnetic particles for each manufacturer. Table 2 below shows the results measured by Nano drop (Thermo scientific, USA).

Example 2: Step of preparing a buffer for cell lysis

2-1: Outline of the experiment

The experiment was conducted while changing the composition of the lysis buffer in order to establish conditions in which the function of efficiently lysing cell membranes, inactivating nucleolytic enzymes, and removing other contaminants works efficiently. Among these constituents, chaotropic reagents are generally referred to as substances that break the bonds of hydrogen ions, and play a role in cell lysis in nucleic acid extraction, unfolding and denature proteins in cells, and nucleic acids and silica. (silica) is known to form a bridge that can be bonded well. In the case of prokaryotes, since it is sufficient to lyse only the cell membrane itself, the composition of a buffer solution for extracting nucleic acids from food poisoning bacteria to be detected was studied. When the cell membrane is dissolved, the nucleic acid is exposed to the nuclease, which requires a substance to protect it. Nucleic acid degrading enzymes use metal divalent ions as co-factors and inhibit their activity with substances that can chelate these metal ions. A typical example is EDTA (Ethylenediaminetetra-acetic acid), and an experiment was conducted to control it. Since changes in pH have an effect on nucleic acids, an experiment was conducted to find the optimal concentration of a substance capable of buffering. The buffer system used in the present invention is based on tris, and it was determined that the buffering effect was good, and an experiment was conducted to control it. In addition, experiments were conducted on additional additives such as detergent.

2-2: Chaotropic reagent

Chaotropic reagent was tested based on guanidine thiocyanate and guanidine hydrochloride, and the concentration was prepared by sequentially diluting from 0.1M to 5.0M. 200 µl of whole blood was added to each 1 ml of reagent, followed by mixing. The tube containing the mixed solution was put on ice and allowed to stand for 10 minutes, and after standing, the tube was mixed well through vortex mixing. 100 µl of magnetic beads were added to this solution, vortex-mixed gently, and then the tube was inserted into a magnetic stand and allowed to stand for 5 minutes. When the solution and the beads were separated, all the solutions were removed, and then washed twice with 80% ethanol (80 ml of 100% ethanol + 20 ml of DDW) solution, and 50 μl of DNA hydration solution was added to make magnetic beads. ) Was released and then inserted into a magnetic stand again, and the solution and beads were separated to recover the solution from which the nucleic acid was eluted.

2-3: Detergent, EDTA, and Tris

Detergent conducted a comparative experiment by mixing Triton X-100 (LPS, Korea), zwitterionic detergent, and CHAPS (Sigma, USA), which are mainly used in nucleic acid extraction reagents, and added EDTA and Tris together to conduct the experiment. The experiment was carried out in the form of mixing each reagent, and the process used for this was carried out in the same manner as the method for experimenting with the chaotropic reagent.

Example 3: Step of selecting conditions for binding genomic DNA and beads in the dissolved solution

In order to check whether the nucleic acid is well bound to the magnetic beads, the binding degree of the nucleic acid was confirmed by comparing the sample with the addition of the magnetic bead and the sample without the addition of the bacterium after dissolving the bacteria. . The bead used was a product of each manufacturer tested above, and was selected as a candidate group bead showing better or similar performance compared to other products with magnetic beads determined after the experiment.

Chaotropic reagent was tested based on guanidine thiocyanate (GTC), and 200 µl of whole blood was added to 400 µl of GTC reagent, respectively, and magnetic beads were added. At this time, magnetic beads were added in an amount between 50-400µl, and the amount of beads that could be optimally bound was determined. In addition, after mixing the lysed blood and beads, the settling time was set to 5 seconds-1 minute, and the tube was inserted into a magnetic stand and allowed to stand for 5 minutes. When the solution and beads were separated, all the solutions were removed, washed twice with 80% ethanol solution, and 50 µl of DNA hydration solution was added to loosen the magnetic beads. The solution was inserted into a magnetic stand again, and the solution and beads were separated to recover the solution from which the nucleic acid was eluted. The amount of nucleic acid extracted under each condition was confirmed using an agarose gel and nano-drop measurement values.

Example 4: Preparation step of washing buffer to remove other contaminants while maintaining target DNA and beads bound

All cells contain various substances, and since only nucleic acids do not bind to beads inserted for nucleic acid extraction, it was necessary to remove other substances attached to the beads to extract only the target nucleic acid. In general, the reagent used in the washing buffer is 70-80% ethanol, and an optimal washing buffer condition was established by controlling isopropanol and salt. To this end, magnetic beads were prepared, 200 µl of the strain cultured in 400 µl of GTC was added thereto, vortex-mixed for 10 seconds, and then allowed to stand at room temperature for 1 minute. After standing, it was mounted on a magnetic stand, and the solution was completely removed after leaving only magnetic beads in the micro-tube. After removing the micro-tube from the magnetic stand, 500 µl each of a washing buffer that combines concentrations of 30-80% ethanol, 25-40% isopropanol, and 0.1M-1.5M NaCl After adding, it was mixed through vortex mixing for 5 seconds. Thereafter, a micro-tube was mounted on a magnetic stand to remove all the remaining solutions other than the beads, and the number of washing was repeated. After removing the remaining washing buffer, it was dried at room temperature for 5 minutes, 50-100 µl elution buffer was added, vortexed for 10 seconds, and then mounted on a magnetic stand. Then, the separated solution was transferred to a new micro-tube and compared through agarose gel electrophoresis and nano-drop measurement values.

Example 5: Step of eluting only DNA from beads

Most of the buffers used to elute nucleic acids are Tris-based reagents. Tris (Tris) is frequently used because it can buffer the eluted nucleic acid, and EDTA, which can inhibit the activity of nucleolytic enzymes, is also widely used. The present inventors generally used a TE buffer (Tris-EDTA buffer) used for eluting nucleic acids. In order to establish the optimal conditions for efficiently eluting DNA from the beads, magnetic beads were prepared, 200 µl of the strain cultured in 400 µl of GTC was added thereto, vortex-mixed for 10 seconds, and then allowed to stand at room temperature for 1 minute. After standing, it was mounted on a magnetic stand, and the solution was completely removed after leaving only magnetic beads in the micro-tube. After removing the micro-tube from the magnetic stand, 500 µl of washing buffer was added at a time, and then mixed through vortex mixing for 5 seconds. Thereafter, a micro-tube was mounted on a magnetic stand to remove all the remaining solutions other than the beads, and the number of washing was repeated once. After removing the remaining washing buffer, dry it at room temperature for 5 minutes, add 100 µl of elution buffer each, settle for 10, 30, 60, 120, 300, 600 seconds, and mount on a magnetic stand. After separating the solution from the beads, the solution from which the nucleic acid was eluted was recovered, and the amount and quality of the nucleic acid according to the standing time were confirmed by agarose gel electrophoresis and nano-drop measurement values.

Example 6: Checking the purity of the eluted nucleic acid and whether the contaminant remains

In order to confirm the purity of the nucleic acid obtained in Example 5, the amount and purity of the nucleic acid were confirmed by checking the wavelength values of 230 nm, 260 nm and 280 nm using a nano-drop spectrophotometer. 2 µl of the extracted nucleic acid-containing solution was collected with a micro-pipette, placed on the measurement position, and measured by pressing the measurement button, and then the value was analyzed. In addition, the extracted solution was electrophoresed on 2 µl and 5 µl agarose gel, and the quality and quantity of nucleic acid were compared with the measured value of nano-drop to confirm the actual quality of nucleic acid.

Example 7: Step of removing residual contaminants and inhibitors of nucleic acid amplification

Polymerase chain reaction was used to confirm the presence or absence of contaminants in the eluted nucleic acid and whether a nucleic acid amplification inhibitor was included. First, the extracted nucleic acids were sequentially diluted to proceed with a conventional polymerase chain reaction, and the presence or absence of amplified nucleic acids was confirmed through agarose gel electrophoresis results. Real-time gene amplification was performed in order to determine whether the exact nucleic acid amplification was inhibited, and the effect on the nucleic acid amplification was confirmed. Detailed reaction reagents and conditions are shown in Table 3 below (real-time polymerase chain reaction conditions and reaction time).

The result of the reaction was checked for the presence or absence of a non-specific band and amplification through agarose gel electrophoresis, and dissolved in order to resolve the conditions in which a substance exhibiting amplification inhibition was found. (lysis) buffer and wash buffer were adjusted. In the case of the Lysis buffer, the concentration of GuHCl and GTC was adjusted between 0.1M-5.0M, the concentration of the surfactant (detergent) solution was changed between 0.1-5.0%, and the concentration of ethanol was changed to 15% in the case of the washing buffer. -It was adjusted between 70%, and the concentration of salt was adjusted between 0.1M-3.0M to ensure removal of inhibitors and contaminants.

Example 8: Extracting nucleic acids against trace contaminated standard strains

In order to apply the above-described nucleic acid extraction method to the nucleic acid extraction of microorganisms contaminated with tissues, the nucleic acid extraction method of the present invention described above is performed by randomly contaminating a standard strain on fish tissues such as flatfish and fish tissue to extract nucleic acids from the target microorganism. We checked whether it can be applied. The contaminated strains are Vibrio spp., known as food poisoning strains, Staphylococcus aureus, Salmonella spp., and Listeria monocytogenes. After inoculating in the medium and incubating OD595 to 0.6, the average CFU of the bacteria that appeared at this absorbance value was calculated, based on this, the extraction of nucleic acids from the number of strains was confirmed, and the Quant-it PicoGreen DNA ds assay kit (life technology) The amount of nucleic acid was quantified through and electrophoresis, and the quality of the nucleic acid was confirmed using real-time gene amplification technique.

Example 9: Step of molecular concentration of extracted nucleic acid for detection of trace nucleic acid

When the number of bacteria to be extracted from nucleic acids is large, the amount of extracted nucleic acids is large and no problem occurs in the next experiment. However, the relative amount of nucleic acid may be insufficient to extract nucleic acids from bacteria contaminated in trace amounts on the sample surface and undergo a process such as polymerase chain reaction. In order to solve this problem, a method of molecularly concentrating the extracted nucleic acid was used. A whole genome amplification (WGA) reaction was performed to molecularly concentrate the extracted nucleic acid in a trace amount. The cultured strains were sequentially diluted to extract nucleic acids, and 1 µl of the extracted nucleic acid was mixed with 1 µl of 1X WGA DB buffer, and allowed to stand at room temperature for 5 minutes. After 5 minutes, 2 µl of 1X WGA NB buffer was added to the mixture and mixed, and 6 µl of WGA SB buffer was added and mixed well. Here, 9 µl WGA RB buffer and 1 µl EM were added and mixed, and the reaction was carried out by controlling the time at 30°C after spinning down. The reaction time was 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 120 minutes, and it was confirmed that there was no problem in the reaction by using the negative control group and the positive control group.

Example 10: Molecular enrichment step for detection of trace nucleic acids using macromolecular crowding effect

To shorten the WGA (Whole Genome Amplification) reaction time, reagents expected to exhibit macromolecular crowding effect were used. As such a reagent, PEG (polyethylene glycol) was used to confirm the effect on the WGA reaction. 1 µl of nucleic acid extracted according to the method described in Example 9 was mixed with 1X WGA DB buffer, and allowed to stand at room temperature for 5 minutes. After 5 minutes, 2 µl 1X WGA RB buffer was added to the mixture, followed by mixing, 6 µl WGA SB buffer and PEG of the corresponding concentration were added and mixed well. Here, 9 µl WGA RB buffer and 1 µl EM were added and mixed, and the reaction was carried out by controlling the time at 30°C after spinning down. The reaction was carried out at reaction times of 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 120 minutes, and it was confirmed that there was no problem in the reaction result by using the negative control group and the positive control group. For the reactants used for amplification, the difference in the amplification amount was confirmed through a real-time gene amplification reaction, and the difference in the amplified amount was confirmed by comparing the control group without the macromolecular crowding reagent and the experimental group.

Example 11: Preparation of molecular diagnostic kit using trace nucleic acids

Molecular diagnosis was performed to diagnose the information of the contaminant through the sample obtained by extracting the nucleic acid and then molecularly enriched. The target bacteria were 10 species as targets, and a total of 10 species including 7 Vibrio strains, 1 Salmonella strain, 1 Staphylococcus aureus, and 1 Listeria were carried out by a molecular diagnosis method using the Taqman probe method. First, the gene information of each strain was obtained from NCBI (National Center for Biotechnology Information), and based on this, a candidate group of gene sites that can be diagnosed for each gene was selected. The following genes were selected as detection candidate genes: hlyA (α-Hemolysin) gene; sdiA (Suppress division inhibitros) gene; spa (S. aureus-specific staphylococcal protein A) gene; vpm (V. parahaemolyticus metalloprotease) gene; tlh (Thermolabile hemolysin) gene; tdh (Thermostable direct hemolysin) gene; Thermostable driect hemolysin-related hemolysin (trh) gene; ctxA (Cholera toxin) gene; epsM (Cholera toxin secretion protein) gene; ftsZ (Filamenting temperature-sensitive mutant Z) gene; rpoB (β-subunit of RNA polymerase) gene; ompU (Outer membrane protein) gene; Invasion protein A (invA) gene; Collagenase gene; 16s rDNA gene; ATPase gene.

After designing each of a primer and a probe at the selected gene site, tests for sensitivity and cross-reactivity were conducted. The information on the oligonucleotide for molecular diagnosis, which was finally confirmed through the test, is shown in Table 4 below.

Primers and probes selected from each candidate group undergo experiments such as limit of detection (LOD) and validation, and control the composition and concentration of reaction reagents for multiplex polymerase chain reaction. Proceeded. The probe for strain detection was FAM, CY5, CalRed fluorescence dye and BHQ quencher dye, and a BioRad CFX96 real-time gene amplification device with 4 fluorescence channels was used. To this end, the diagnostic kit was divided into SET A and SET B, and SET A detects Vibrio and Salmonella, and SET B detects Listeria and Staphylococcus. In each kit, as an internal control, a control template made from the lentinula gene, a primer, and a probe set were added together to increase the accuracy of the result analysis.

Example 12: Comparison of detection effect according to extracted nucleic acid and molecular concentration

Using 10 pg of nucleic acid extracted by the above nucleic acid extraction method from the standard strain of the food poisoning causative bacteria, it is used as a PCR template for detection kits SET A and SET B, and the WGA kit (SEQ-TempliGenTM WGA) sold by Solgent Co., Ltd. kit) was used as a PCR template by reacting 10 pg of nucleic acid for 1 hour as a normal WGA sample, and the enhanced WGA was used as a PCR template after 1 hour reaction using 10 pg of nucleic acid using the WGA method developed in the present invention. Was used. The amount of extracted nucleic acid in each reaction was equal to 10 pg, and the detection time was shortened for each reaction by molecular concentration, and the effect of the amplified fluorescence value and Ct value was confirmed. As shown in FIGS. 18 and 19, the detection time was shortened by molecular concentration, and in particular, in the case of enhanced WGA, the detection time was maximally shortened, and thus it was confirmed that detection in trace amounts of nucleic acids can be made easier.

Example 13: Application of UDG (Uracil DNA glycosilase) system to prevent contamination

In order to prevent cross contamination by repeated experiments, a UDG (Uracil DNA glycosilase) system that recognizes and cuts uracil DNA was applied to the reaction buffer. In order to find the conditions in which UDG does not affect the PCR reaction and effectively removes the contaminated previous test product, a PCR template was prepared using dNTP containing uracil, and it was artificially mixed with the actual reaction solution. It was confirmed that it was removed from the solution, and that it did not affect the actual PCR through real-time PCR, and the contaminated PCR product was removed through agarose gel electrophoresis, and only the sequence to be amplified was amplified. It was confirmed to be.

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Solgent Co., Ltd. <120> Method for Detection of Food Poisoning Bacteria By Using Gene Amplification and Kit for Use in The Same Method <130> PN150548 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 1 gacgacgtcc agctaataac g 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 2 cagatgctaa caaagctcaa gc 22 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 3 cagaaactgg tgaagaaaat ccattcatcg gta 33 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 4 tggtggaaac acgccaagg 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 5 actgcattag gagcgccac 19 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 6 accaatgact gttgccactg ttcaaactcg 30 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 7 cagtgaaggg aaaatgcaag aagaag 26 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 8 ccaagcgctt gcaactgctc tttag 25 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 9 tttgccgaaa aatctggaag gtcttgtagg t 31 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 10 cgtcgatctt agttgctaca ccg 23 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 11 caaacttccg tacttacatc tcttacaac 29 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 12 atgattgatg ctggcgatgt attgggtaaa gtt 33 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 13 gcgtaatccg aaacgctgg 19 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 14 cgatcgatcc ggtattaaag c 21 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 15 aatttcaggc agggtcattt acattgggat g 31 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 16 cgacggacat cgacagacg 19 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 17 cagccttgac ccggaagc 18 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 18 taatttgatg gatctcatta cacttaagtt gg 32 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 19 gtgagtacag cggccgtgtc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 20 cggttcggga ccaaatcgca 20 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 21 aaattgcttc gaatcggttc ggccatcata 30 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 22 cggtaaccaa tgcttccaaa tc 22 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 23 caaccaaccg caactctccc 20 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 24 agccctccaa ggcaactcga gtacag 26 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 25 cgctacgacc tgtctacgg 19 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 26 agcttcttca tcacttcgac g 21 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 27 tgtttcgtca agagtgcctt gctcttca 28 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 28 ccgagcattt gtgtaagttc ctcttt 26 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 29 cgcgcgcaac tgcattatga tttac 25 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 30 tcgagcgcgt aaagcaatgg aagaatattt aga 33 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 31 gagggaaacc tgccaatcc 19 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 32 cttggagcat tcccacaacc 20 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 33 agcttggagg gaagagccgt gga 23 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 34 gttgggttaa gtcccgcaac g 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for real time PCR <400> 35 ttatcaccgg cagtctccct g 21 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Probe for real time PCR <400> 36 acccttatcc ttgtttgcca gcgagtaat 29

Claims (5)

A kit for real-time polymerase chain reaction for detection of Vibrio spp. Or Salmonella spp. Bacteria comprising the following primer-probe set:
A second primer-probe set consisting of SEQ ID NOS: 4, 5 and 6;
A fourth primer-probe set consisting of SEQ ID NOS: 10, 11, and 12;
A fifth primer-probe set consisting of SEQ ID NOS: 13, 14 and 15;
A sixth primer-probe set consisting of SEQ ID NOS: 16, 17 and 18;
A seventh primer-probe set consisting of SEQ ID NOS: 19, 20 and 21;
An eighth primer-probe set consisting of SEQ ID NOS: 22, 23 and 24;
A ninth primer-probe set consisting of SEQ ID NOS: 25, 26 and 27;
A tenth primer-probe set consisting of SEQ ID NOS: 28, 29 and 30;
An eleventh primer-probe set consisting of SEQ ID NOS: 31, 32, and 33; And
A twelfth primer-probe set consisting of SEQ ID NOS: 34, 35 and 36.
The method of claim 1, wherein the Vibrio bacterium belonging to the genus Vibrio are para to Morley tea kusu (V. parahaemolyticus), Vibrio flu via-less (V. fluvialis), Vibrio know fun tea kusu (V. alginolyticus), Vibrio vulnificus (V . vulnificus), Vibrio insignificant kusu (V. mimicus), Vibrio cholerae (V. cholerae), and Vibrio meth struck Cove (V. metschnikov) one or more selected from the group consisting of bacteria belonging to the genus Vibrio is, real-time polymerase chain reaction kit for .
The real-time PCR reaction kit according to claim 1, wherein the real-time PCR reaction kit further comprises UDG (uracil DNA glycosylase).
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