KR20170078463A - A method and test kit for detecting microorganisms using pcr product - Google Patents

Abstract

The present invention relates to a microorganism detection method and a microorganism detection kit using a polymerase chain reaction amplification product, and a method for rapidly detecting various microorganisms and a detection kit using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for detecting a microorganism using a polymerase chain reaction amplification product and a method for detecting a microorganism using the PCR product,

The present invention relates to a microorganism detection method and a microorganism detection kit using a polymerase chain reaction amplification product, and provides a method for rapidly detecting various microorganisms including food poisoning causing microorganisms.

Food poisoning is a disease accompanied by symptoms such as fever, nausea, vomiting, diarrhea and abdominal pain. Most of them are bacterial food poisoning. Bacterial food poisoning is classified as infectious or toxic according to the onset type.

Infection type of food poisoning is to develop and proliferate within Chapter microorganisms by ingestion of food contaminated with pathogenic microorganisms, Salmonella (Salmonella spp), Vibrio para Molly Tee hee Caicos (Vibrioparaheamolyticus), E. coli O157 (Escherichia coli O157: H7 ) are the main causative bacteria.

Poisonous food poisoning can occur by ingesting toxins produced by pathogenic microorganisms that multiply in food. In this case, the pathogens can already be caused by the presence of death, even if the toxins within foods, Staphylococcus aureus (Staphylococcus aureus), Clostridium botyul rineom (Clostridiumbotulinum) is the major causative agent.

In food poisoning, the rate of incidence is increasing due to changes in lifestyle patterns and global warming, such as the expansion of group meals or the increase in eating out, and the scale is also becoming larger and larger, so it is important to detect causative organisms early.

Nevertheless, pathogenic microorganisms including food poisoning bacteria are usually detected by classical culture method, and this method can not guarantee rapid detection results because it takes a long period of incubation and analysis period.

In order to overcome the above problem, the enzyme polymerization reaction (PCR) is applied. However, the electrophoresis method has to be performed in order to determine whether it is positive. In addition, even if the enzymatic polymerization reaction product is identified, a false positive due to a non-specific reaction may occur and a long additional procedure such as Southern hybridization may be used to determine the false positive Required.

Therefore, a side-view flow immunoassay, which is capable of detecting microorganisms at low cost and low in cost, has been developed. However, in order to obtain an assayable cell mass (10 ⁵ cfu / mL or more) with low sensitivity, There is a problem in that practicality is low.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a microorganism detection method and a microorganism detection kit.

(A) preparing a second primer hybridizing specifically to a gene of a target microorganism and bound to a first primer and a second labeling substance to which the first labeling substance 202 is bound; (b) performing a polymerase chain reaction (PCR) using the first primer and the second primer; And (c) performing an immunoassay using a probe that specifically binds to the nucleic acid sequence amplified by the polymerase chain reaction.

In one embodiment, after the step (b), the antisense oligomer of the first primer, the antisense oligomer of the second primer, or the S1 nucleic acid hydrolase is added to the polymerase chain reaction product before the step (c) And then reacting the reaction mixture.

In one embodiment, in step (a), the first primer and the second primer may be bound to a labeling substance at a 3 'end, a 5' end, or a predetermined position.

In one embodiment, the labeling substance in step (a) is selected from the group consisting of biotin, digoxigenin, dinitrophenol, aminoacetylfluorene, sulfonate, Fluorescein isothiocyanate Aminomethylcoumarin, FAM (Carboxyl fluorescein-aminohexyl-amidite), TAMRA (Tetramethyl-6-Carboxyrhodamine) and cyanine.

In one embodiment, in the step (c), the probe may include a reactive substance that specifically binds to the first labeling substance, and a coloring particle.

In one embodiment, the coloring particles may be selected from the group consisting of latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, and magnetic particles.

In one embodiment, the step (c) comprises contacting a result of the polymerase chain reaction with a probe that specifically binds the first labeling substance and a second labeling substance immobilized reaction membrane ); And determining the presence or absence of the microorganism through the color development reaction of the probe.

According to another aspect of the present invention, there is provided a method for detecting a target microorganism, comprising: a first solution comprising a first primer to which a first labeling substance is bound and a second primer to which a second labeling substance is bound; And a diagnostic strip on which a sample pad for dropping a microorganism sample, a conjugation pad including a probe, and a test membrane on which a test line is formed are sequentially arranged Thereby providing a microorganism detection kit.

In one embodiment, the microorganism detection kit may further comprise a second solution comprising an antisense oligomer of the first primer, an antisense oligomer of the second primer, or a S1 nucleic acid hydrolase (S1 nuclease).

In one embodiment, the probe may include a reactive substance that specifically binds to the first labeling substance, and a coloring particle.

In one embodiment, the coloring particles may be selected from the group consisting of latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, and magnetic particles.

In one embodiment, the anti-second marker substance antibody 107 may be immobilized on the test line.

In one embodiment, the test membrane may further comprise a control line on which the second antibody that does not specifically react with the first labeling substance and the second labeling substance is immobilized.

The microorganism detection method and the microorganism detection kit according to one aspect of the present invention have improved portability as compared with existing microorganism detection methods and can be applied immediately on site and can detect various microorganisms quickly and minimize false positives .

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

FIG. 1 illustrates a diagnostic strip according to an embodiment of the present invention.
FIG. 2 illustrates a method of detecting a microorganism according to an embodiment of the present invention.
Figure 3 is a schematic representation of a dimer formed by the interaction between a first primer and a second primer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly explain the present invention, parts not related to the description are omitted, and like parts are denoted by similar reference numerals throughout the specification.

When an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the skill of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. It is not intended that the invention be limited to the particular methodology, protocols, and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, 5 'to 3', amino acid sequences from left to right, amino to carboxyl.

Hereinafter, the present invention will be described in more detail.

Microorganism detection method

The method of detecting microorganisms according to one aspect of the present invention comprises the steps of (a) hybridizing specifically to a gene of a target microorganism and comprising a first primer 201 and a second labeling substance 302 to which a first labeling substance 202 is bound Preparing a combined second primer (301); (b) performing a polymerase chain reaction (PCR) using the first primer 201 and the second primer 301; And (c) performing immunoassay using a probe that specifically binds to the nucleic acid sequence amplified by the polymerase chain reaction.

The microorganism detection method can effectively detect a target microorganism by combining a polymerase chain reaction and an immunoassay. The microorganisms include viruses, bacteria, actinomycetes, protozoa, yeast, fungi, mushrooms, algae, ricketts, undifferentiated cells and tissue cultures of animals and plants, and generally have microscopic organisms smaller than 0.1 mm . ≪ / RTI > In particular, the microorganisms may include various types of hospital microorganisms that can cause food poisoning.

In step (a), a pair or more than one pair of primers hybridizing specifically to the gene of the target microorganism may be prepared, and the labeling substance may be bound to the primer so that the primer can be easily identified by visual observation or other means .

The primer may be a short nucleic acid sequence including a free 3 'hydroxyl group and form a base pair with a template of the complementary nucleic acid, and a strand copy of the nucleic acid template Can be used as a starting point. The primer can initiate DNA synthesis in the presence of a reagent for polymerization and a different four nucleoside triphosphate at the appropriate buffer solution and temperature and hybridize with the nucleic acid present in the target microorganism to produce a specific gene of the target microorganism Can be used for amplification.

In consideration of the efficiency of amplification, the primer may preferably be a single strand and may be a deoxyribonucleotide. The primers may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides. The primers include, but are not limited to, skeletal modified nucleotides such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and a base Nucleotides with modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethytyl-, propynyl 7-deazapurines having C-7 substituents (the substituents are fluoro, bromo, chloro, iodo, -, Methyl-, ethyl-, -, formyl -, alkynyl -, alkenyl-, thiazol crude reel-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine.

The hybridization means that two single-stranded nucleic acids form a duplex structure by pairing complementary base sequences. The hybridization may occur in a perfect match between single stranded nucleic acid sequences or in the presence of some mismatching bases. The degree of complementarity required for the hybridization may vary depending on the hybridization reaction conditions, and can be controlled by temperature, in particular. Generally, when the hybridization temperature is high, hybridization is likely to occur when the hybridization is complete, and hybridization may occur if the hybridization temperature is low, even if some mismatch exists. Therefore, as the hybridization temperature is lowered, the degree of mismatch, in which hybridization may occur, may increase.

In one embodiment, the pair of primers may be prepared to hybridize to a characteristic gene of the microorganism so as to determine presence or absence of the target microorganism. When the target microorganism is present in the sample, Since the nucleotide sequence of the characteristic gene is heavy, the presence or absence of the microorganism can be determined through the amplified product.

In the step (b), the polymerase chain reaction (PCR) may be performed according to a general reaction condition using a conventional PCR instrument known in the art, or may be carried out with some modifications. In the microorganism detection method, the DNA of the characteristic gene may be amplified using the primer pair, and then the DNA may be amplified. The amplified DNA may be easily identified by immunoassay.

Meanwhile, since the primer pair includes the labeling substance bound to the primer, the DNA amplified by the polymerase chain reaction can be easily identified by the labeling substance.

The labeling substance is a substance which generates a signal that can be sensed by the naked eye or a sensor. Examples of the labeling substance include biotin, digoxigenin, dinitrophenol, aminoacetylfluorene, Aminomethylcoumarin, FAM (Carboxyl fluorescein-aminohexyl-amidite), TAMRA (Tetramethyl-6-Carboxyrhodamine), and cyanine (Cyanine) in the group consisting of FITC (Fluorescein isothiocyanate), rhodamine, aminomethylcoumarin Or more, but the kind thereof is not particularly limited as long as it can function as a cover.

The first primer 201 and the second primer 301 may be composed of a first label material 201 and a second primer 301. The first primer 201 and the second primer 301 may be composed of a first label material 202 And a second labeling material 302, respectively. In one embodiment, the first primer 201 and the second primer 301 may be bound to a labeling substance at a 3 'end, a 5' end, or a predetermined position.

In the step (c), immunoassay may be performed using a probe that specifically binds to the nucleic acid sequence amplified by the polymerase chain reaction. The immunoassay means an assay for detecting or quantifying an analyte, such as a nucleic acid of interest, comprising an immune response between the antibody and the antigen.

Typically, the immunoassay can be performed by using a probe 400 that can hybridize to a target nucleic acid molecule in a sample. In addition, the immunoassay can be achieved by using an antibody specific to the hybrid formed by the amplified nucleic acid sequence and the hybridization of the probe (400).

In one embodiment, step (c) can be performed by lateral flow immunoassay, and the presence or absence of the labeling substance can be quickly and accurately grasped by the method.

Specifically, in the step (c), a probe that specifically binds the polymerase chain reaction product to the first labeling substance 202 and a reaction membrane to which the anti-second labeling substance antibody 107 is immobilized to a test membrane (103); And determining the presence or absence of the microorganism through the color development reaction of the probe 400.

FIG. 1 is a schematic diagram of a diagnostic strip 100 according to an embodiment of the present invention, and FIG. 2 is a schematic view of a microorganism detection method according to an embodiment of the present invention.

Referring to FIGS. 1 and 2, in step (c), a probe that specifically binds the polymerase chain reaction product to the first labeling substance 202 and an anti-second labeling substance antibody 107 Can be diffused in a fixed test membrane 103 and the amplified DNA can be fixed to the reaction membrane 103 and confirmed by the probe 400. [

The probe 400 may bind to or hybridize with the DNA amplified by the polymerase chain reaction to indicate presence or absence of the target microorganism. The type of the probe 400 is not particularly limited as long as it can display the presence or absence of the DNA by a specific interaction as a substance that specifically binds to the amplified DNA.

In one embodiment, the probe 400 may include a reactive substance 401 that specifically binds to the first labeling material 202, and chromogenic particles 402. That is, since the amplified DNA includes the first labeling substance 202 and the second labeling substance 302 at both ends or a predetermined position, the reaction membrane 103 And the presence or absence of the amplified DNA can be easily identified because the probe 400 binds to the first labeling material 202.

Since the probe 400 includes the reactive substance 401 and can specifically bind to the first labeling substance 202 and includes the coloring particles 402, It is possible to easily display whether or not the first labeling material 202 is bonded.

The coloring particles 402 may be selected from the group consisting of latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers and magnetic particles, but they may be easily identified by visual inspection or other means Materials that can be used are not particularly limited.

That is, the probe 400 may include particles that are colored by binding or aggregation so that the probe 400 can be visually recognized. If the probe 400 is visually confirmed by color development, The presence can easily be identified.

On the other hand, the primer may include one or more pairs of primers.

In one embodiment, the primer may include a pair of primers composed of a first primer 201 and a second primer 301, and a pair of primers composed of a third primer and a fourth primer .

For example, the first primer 201 and the second primer 301 may include a first labeling material 202 and a second labeling material 302, respectively, which are different from each other, and the third primer and the fourth primer May include a first labeling material 202 and a third labeling material, respectively.

That is, the microorganism detection method can use a primer that is specifically hybridized to a characteristic gene of a target microorganism. However, detection accuracy with respect to the target microorganism can be further improved by using more than one pair of primers.

After the step (b), the antisense oligomer of the first primer 201, the antisense oligomer of the second primer 301, or the S1 nucleic acid hydrolysis And reacting the enzyme (S1 nuclease).

That is, the microorganism detection method uses the first primer 201 to which the first labeling substance 202 is bound and the second primer 301 to which the second labeling substance 302 is bound, And amplification can be quickly confirmed by immunoassay. However, when the first primer 201 and the second primer 301, which are not hybridized to a specific gene after the polymerase chain reaction, are mutually reacted with each other, If combined, it can lead to false positives.

FIG. 3 is a schematic diagram of a dimer formed by the interaction between the first primer 201 and the second primer 301.

Referring to FIG. 3, the microorganism detection method can identify amplification of DNA through a labeling substance bound to primers at both ends of the amplified DNA. However, in the present invention, 1 primer 201 and second primer 301 can form a dimer due to intermolecular interaction such as hydrogen bonding.

Since the primer forming the dimer includes both the first labeling material 202 and the second labeling material 302, it is preferable that the primer binds to the anti-second labeling material antibody 107 like the amplified DNA, (400). ≪ / RTI > In this case, even when the target microorganism is not present in the sample and the DNA of the microorganism is not amplified, a positive reaction can be induced.

Therefore, when the antisense oligomer of the first primer 201 and the antisense oligomer of the second primer 301 are reacted after the polymerase chain reaction is completed, the first primer 201 and the second primer 301) can not form a dimer, so that the false positives can be minimized.

In addition, when the S1 nucleic acid is reacted after the PCR is completed, the first primer 201 and the second primer 301 which are not hybridized in a single strand are decomposed Since the dimer can not be formed, the false positives can be excluded.

Microbial detection kit

The microorganism detection kit according to another aspect of the present invention comprises a first primer 201 to which a first labeling substance 202 is bound and a second labeling substance 302 to which a second labeling substance 302 is bound, 2 primer (301); A sample pad 101 for dropping a microorganism sample, a conjugation pad 102 including a probe 400 and a test membrane 103 having a test line 104 formed thereon, And a diagnostic strip 100 arranged in this order.

The first solution is hybridized specifically to the gene of the target microorganism and the first primer 201 to which the first labeling substance 202 is bound and the second primer 301 to which the second labeling substance 302 is bound .

The diagnostic strip 100 includes a sample pad 101 for dropping a sample of microorganisms, a conjugation pad 102 including a probe 400, and a reaction membrane 104 having a test line 104 formed thereon. (test membrane) 103 may be sequentially arranged.

The sample pad 101 may serve as an inlet through which the sample is directly introduced. The sample pad 101 may filter a solid component contained in the sample as a porous material and transfer the liquid component to the reaction membrane 103 . The sample pad 101 separates a solid component contained in the sample and unnecessary impurities from the sample, and thus may be an adsorbent material.

The probe 400 may be condensed with a conjugate pad 102 that specifically binds to the nucleic acid sequence amplified by the polymerase chain reaction and the probe 400 may be conjugated to the first labeling substance A reactive substance 401 that specifically binds to the coloring particles 402, and a coloring particle 402.

Since the condensation pad 102 is located downstream of the sample pad 101, when the microorganism sample loaded on the sample pad 101 moves via the condensation pad 102, The probes 400 can be specifically coupled. That is, when the microorganism sample contains a large amount of characteristic DNA of the target microorganism by the polymerase chain reaction, the probe 400 condensed in the condensation pad 102 can be specifically detected with the first labeling substance 202 And can bind to the characteristic DNA.

The test membrane 103 may include the immobilized anti-second marker substance antibody 107, and may be a nylon, a polyester, a cellulose, a polysulfone, a polyvinyl Cellulose acetate, polyurethane, glass fiber, and nitrocellulose may be used, but the kind thereof is not particularly limited. A test line 104 is formed on the downstream side of the reaction film 103 and the inspection line 104 may include the immobilized anti-second labeling material antibody 107.

That is, the DNA in the microorganism sample bound to the probe 400 via the condensation pad 102 may be bonded to the inspection line 104 positioned downstream and be visually or otherwise identified.

In one embodiment, when the first labeling material 202 is biotin, the condensation pad 102 may be formed of streptavidin and gold particles that specifically bind to the biotin And the probe 400 may be coupled with the first label material 202 bound to a predetermined position of the DNA so that the probe 400 may be visually confirmed by the inspection line 104 positioned at the downstream side .

The test membrane 103 may include a control line on which the second antibody 108 that does not specifically react with the first labeling substance 202 and the second labeling substance 302 is immobilized. 105).

Various methods capable of ensuring detection reliability can be applied to the reference line 105. For example, the control line 105 may include a separate antibody that does not specifically react with the first labeling substance 202 and the second labeling substance 302, If the same color is developed, the user can predict the possibility of error in the test result.

The microbial detection kit may further include an absorbent pad 106, which may be located at the distal end of the diagnostic strip 100. The absorbent pad 106 absorbs excessive sample or sample developing solution to prevent disturbance of the detection result, and can be applied to any material having excellent absorbency such as cotton, absorbent gel, and the like.

The microorganism detection kit may further comprise a second solution comprising an antisense oligomer of the first primer 201, an antisense oligomer of the second primer 301, or a S1 nucleic acid hydrolase (S1 nuclease).

The second solution may be added to the reaction product after the polymerase chain reaction and reacted for a predetermined period of time. By this reaction, dimer formation between the first primer 201 and the second primer 301 is inhibited, The false positives can be minimized.

It will be apparent to those skilled in the art that various changes and modifications may be made thereto without departing from the spirit and scope of the present invention as set forth in the appended claims. It is encompassed in the technical idea of.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Production Example 1: Preparation of a primer having a labeling substance bound thereto

Primers hybridizing to genes characteristic of specific microorganisms were prepared as shown in Table 1 below. DIG means digoxigenin, and BTN means biotin. Biotin was attached to the 5 'end of the first primer 201 as the first labeling substance 202 and digoxigenin was attached to the 5' end of the second primer 301 as the second labeling substance 302.

Target microorganism (gene name) Primer sequence Salmonella
( hto gene) 5 'BTN-ATG TTG TCC TGC CCC TGG TAA GAGE
5 'DIG-ACT GGC GTT ATC CCT TTC TCT GGT G Vibrio
( tlh gene) 5 'BTN-GCT ACT TTC TAG CAT TTT CTC TGC
5 'DIG-AAA GCG GAT TAT GCA GAA GCA CTG Staphylococcus aureus
( nuc gene) 5 'BTN-CAA GCC TTG ACG AAC TAA AGC
5 'DIG-GCG ATT GAT GGT GAT ACG G Listeria monocytogenes
( inline gene) 5 'BTN-CAA ATT TGT TAA AAT CCC AAG TGG
5 'DIG-ACT ATC TAG TAA CAC GAT TAG TGA E. coli O157: H7
( stx 2 gene) 5 'BTN-TCG CCA GTT ATC TGA CAT TCT G
5 'DIG-GGC ACT GTC TGA AAC TGC TCC

Production Example 2: Preparation of a probe

Streptavidin (streptoavidin) and gold particles were combined to prepare a probe.

Streptavidin was diluted with 0.5 x PBS (phosphate buffered saline, PBS) at a concentration of mg / ml to prepare the above probe. The diluted solution was added to a gold particle solution (pH 7.1) having a diameter of 40 nm having an absorbance (540 nm) of 15 to a final concentration of 40 μg / ml, followed by slow stirring at room temperature for 5 minutes. Finally, the concentration of bovine serum albumin (BSA) was adjusted to 1%, and the reaction was continued for 5 minutes. The prepared streptavidin-gold particle conjugate (probe 400) was stored in a refrigerator at about 4 ° C. until use.

Preparation Example 3: Preparation of a diagnostic strip

The streptavidin-gold particle conjugate (probe) prepared in Preparation Example 2 was mixed with a PBS solution containing 6% sucrose in a ratio of 2: 1, the mixture was uniformly wetted with polyester fiber, Pad.

The anti-digoxigenin antibody was diluted with 0.1 M phosphate buffered saline (PBS) to a concentration of 1 mg / ml. The nitrocellulose pad (width 25 mm, pore size 10-12 μm) To the inspection line position.

The anti-rabbit immunoglobulin G antibody obtained by immunizing rabbit immunoglobulin G with the mouse was diluted to a concentration of 1 mg / ml with 0.1 M PBS, sprayed onto the control line 105 of the nitrocellulose pad, and incubated at 37 ° C in a thermostat Dried and fixed.

PBS containing 0.05% by weight of bovine serum albumin, 4% by weight of sucrose and 0.0625% by weight of ionic surfactant was sprayed on the remaining margins of the nitrocellulose pad except for the fixed portion of the antibody, Lt; 0 > C thermostat for 60 to 120 minutes.

The fabricated condensation pad and nitrocellulose pad were attached to an adhesive coated polypropylene plate backing plate, and an absorbent pad was attached downstream of the nitrocellulose pad attached to the support.

Experimental Example 1: Polymerase chain reaction (PCR) was carried out

The food contaminated with the Salmonella strain was pulverized and added to the 10-fold weight of the pre-culture solution and homogenized.

The liquid culture component was separated from the pre-culture solution through a filter. The liquid component was allowed to stand at room temperature for a certain time, and polymerase chain reaction was performed using the primer sequence of Preparation Example 1.

The polymerase chain reaction was followed by the method of Sambrook and Russell (2001). The polymerase chain reaction was carried out in the presence of 1 μl of the separated liquid components, 5 μl of 10 × PCR reaction buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 3 μl of MgCl 2 solution (final concentration, 1 μl of PCR primer, 1 μl of BSA (20 mg / ml, 1 μl), 1 μl (20 pmoles) of the PCR DIG labeling mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 1.9 mM dTTP, Bovine Serum Albumin, Roche, Germany), and 32 μl of distilled water was added to 0.5 μl (2.5 U) of Taq DNA polymerase (Roche, Germany).

After the initial denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 1 min, annealing at 53.2 ° C for 1 min and extension at 72 ° C for 2 min were repeated 30 times and finally at 72 ° C for 10 min Lt; / RTI > PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Calif. USA) as a thermocycler.

Experimental Example 2: Evaluation using a lateral flow diagnostic strip

The polymerase chain reaction product of Experimental Example 1 was added to the diagnostic strip of Preparation Example 3 and developed. The results of the polymerase chain reaction showed a color reaction in the test line, but no color reaction was observed in the control line, so that the contamination by Salmonella strains could be visually confirmed.

As a result of repeating the experiment while varying the amounts of the liquid components or microorganisms isolated in Experimental Example 1, the microorganisms were identified in a short time even by a very small amount of the samples.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

100: diagnostic strip
101: sample pad
102: condensation pad
103: reaction membrane
104: Inspection line
105: Taekwondo
106: Absorption pad
107: anti-second marker substance antibody
108: Second antibody
201: First primer
202: First label substance
301: Second primer
302: Second labeling substance
400: probe
401: Reactive material
402: coloring particles

Claims (13)

(a) preparing a second primer hybridizing specifically to a gene of a target microorganism and having a first primer and a second label attached thereto, to which a first label is bound;
(b) performing a polymerase chain reaction (PCR) using the first primer and the second primer; And
(c) performing an immunoassay using a probe that specifically binds to the nucleic acid sequence amplified by the polymerase chain reaction. The method according to claim 1,
After the step (b), the antisense oligomer of the first primer, the antisense oligomer of the second primer, or the S1 nucleic acid hydrolase (S1 nuclease) is reacted to the polymerase chain reaction product before the step (c) Further comprising the step of: The method according to claim 1,
Wherein the first primer and the second primer are bound to a labeling substance at a 3 'end, a 5' end, or a predetermined position in the step (a). The method according to claim 1,
In step (a), the labeling substance may be selected from the group consisting of biotin, digoxigenin, dinitrophenol, aminoacetylfluorene, sulfonate, FITC (fluorescein isothiocyanate), rhodamine ), Aminomethylcoumarin, Carboxy fluorescein-aminohexyl-amidite (FAM), TAMRA (Tetramethyl-6-Carboxyrhodamine) and cyanine. The method according to claim 1,
Wherein the probe comprises a reactive substance that specifically binds to the first labeling substance, and a coloring particle in the step (c). 6. The method of claim 5,
Wherein the coloring particles are at least one selected from the group consisting of latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers and magnetic particles. The method according to claim 1,
(C) diffusing the result of the PCR reaction on a test membrane immobilized with a probe and an anti-second labeling antibody specifically binding to the first labeling substance; And
And determining the presence or absence of the microorganism through the color development reaction of the probe. A first solution comprising a first primer to which a first labeling substance is bound and a second primer to which a second labeling substance is bound, the first solution being hybridized specifically to a gene of the target microorganism; And
A diagnostic strip comprising a sample pad for dropping a microorganism sample, a conjugation pad including a probe, and a test membrane on which a test line is formed are sequentially arranged. Detection kit. 9. The method of claim 8,
Further comprising a second solution comprising an antisense oligomer of said first primer, an antisense oligomer of said second primer, or S1 nucleic acid hydrolase (S1 nuclease). 9. The method of claim 8,
Wherein the probe comprises a reactive substance that specifically binds to the first labeling substance, and chromogenic particles. 11. The method of claim 10,
Wherein the coloring particles are at least one selected from the group consisting of latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers and magnetic particles. 9. The method of claim 8,
Wherein the test line is immobilized with the anti-second marker antibody. 9. The method of claim 8,
Wherein the test membrane further comprises a control line immobilized with a second antibody that does not specifically react with the first labeling substance and the second labeling substance.

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