KR20170078456A - A method and test kit for detecting microorganisms using lamp product - Google Patents

Abstract

The present invention relates to a microorganism detection method and a microorganism detection kit using the loop-mediated isothermal amplification 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 loop-mediated isothermal amplification reaction product and a method for detecting a microorganism using the loop-

The present invention relates to a microorganism detection method and a microorganism detection kit using a loop-mediated isothermal amplification reaction 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 method of a diagnosis kit method capable of detecting microorganisms at low cost and low cost of testing has been developed. However, in order to obtain a cell mass (10 ⁵ cfu / mL or more) 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.

An aspect of the present invention provides a method for detecting a target microorganism, comprising the steps of: (a) preparing a primer hybridizing specifically to a gene of a target microorganism and bound to a first marker substance; (b) performing a Loop-mediated Isothermal Amplification (LAMP) using the primer; And (c) performing immunoassay using a first probe and a second probe that specifically bind to the nucleic acid sequence amplified by the loop-mediated isothermal amplification reaction. to provide.

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

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 first probe may include an oligonucleotide that specifically binds to the gene of the target microorganism, and a second marker substance.

In one embodiment, step (c) comprises reacting the loop-mediated isothermal amplification reaction product with the first probe; Diffusing the reactant into a test membrane which is sequentially immobilized with a second probe and an anti-first labeling substance antibody specifically binding to the second labeling substance; And determining the presence or absence of the microorganism through the color reaction.

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

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 (b) is followed by the step of reacting the antisense oligomer of the primer or the S1 nucleic acid hydrolase (S1 nuclease) with the loop-mediated isothermal amplification reaction product before the step (c). As shown in FIG.

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

In addition, since the loop-mediated isothermal amplification reaction is applied to the microorganism detection method according to one aspect of the present invention, the amplification efficiency is excellent, and there is no need to precisely change the temperature, so expensive equipment is unnecessary.

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 interaction between a primer and a first probe.

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

(A) preparing a primer 201 hybridized specifically to a gene of a target microorganism and to which a first labeling substance 202 is bound; (b) performing a Loop-mediated Isothermal Amplification (LAMP) using the primer 201; And (c) performing immunoassay using a first probe 300 and a second probe 400 that specifically bind to the nucleic acid sequence amplified by the loop-mediated isothermal amplification reaction; And a method for detecting microorganisms.

The microorganism detection method can effectively detect the target microorganism by combining loop-mediated isothermal amplification reaction and 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 the step (a), one or more primers 201 that hybridize specifically to a gene of the target microorganism may be prepared. The primer 201 may include a labeling substance so that the primer 201 can be easily identified.

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 201 can initiate DNA synthesis in the presence of a reagent for polymerization and a different four nucleoside triphosphate at a suitable buffer solution and temperature and is capable of hybridizing with a nucleic acid present in the target microorganism, Can be used to amplify specific genes.

In consideration of the efficiency of amplification, the primer may preferably be a single strand and may be a deoxyribonucleotide. The primer 201 may comprise naturally occurring dNMPs (i.e. dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer 201 may also include ribonucleotides. The primer 201 may comprise a framework-modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, 2'-O-methyl RNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA , And nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, , 7-deazapurines having a C-7 substituent (the substituents are fluoro, bromo, chloro, iodo, iodo, -, methyl-, ethyl-, Vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.

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 primer 201 may be prepared to hybridize to a characteristic gene of the microorganism so as to determine the presence or absence of the target microorganism. When the target microorganism is present in the sample, a loop- The nucleotide sequence of the characteristic gene is amplified by the amplified product, so that the presence of the microorganism can be determined through the amplicon.

In the step (b), the loop-mediated isothermal amplification reaction is similar to the conventional polymerase chain reaction (PCR), but the conventional polymerase chain reaction is repeatedly performed three steps of denaturation, The target gene is amplified, so that it is necessary to continuously change the temperature during the reaction. On the other hand, the isothermal amplification reaction can be performed at a constant temperature. Since the loop-mediated isothermal amplification reaction uses a Bst DNA polymerase having an exonuclease activity of a nucleic acid instead of a Taq DNA polymerase, it can induce the denaturation of double helix structure of DNA without being dependent on heat .

Since the isothermal amplification reaction does not involve a temperature change during the amplification of the gene, it is possible to amplify the gene at a constant temperature without expensive professional equipment, and the amplification efficiency is excellent.

The microorganism detection method can efficiently amplify a target microorganism gene through the isothermal amplification reaction, and can quickly detect a target microorganism through an inexpensive and miniaturized apparatus.

Meanwhile, since the primer 201 includes the labeling substance bound to the primer 201, the DNA amplified by the loop-mediated isothermal amplification reaction can be easily identified by the labeling substance.

The labeling substance can be identified by visual inspection, a sensor or other means, and can be, for example, biotin, digoxigenin, dinitrophenol, aminoacetylfluorene, sulfonate, FITC One or more selected from the group consisting of fluorescein isothiocyanate, rhodamine, aminomethylcoumarin, FAM (Carboxyl fluorescein-aminohexyl-amidite), TAMRA (Tetramethyl-6-Carboxyrhodamine) and cyanine However, the type thereof is not particularly limited as long as it can function as a cover.

Specifically, the primer 201 may be one or more of the loop-mediated isothermal amplification reactions, and at least one of the primers 201 may include the first label material 202. At this time, the primer 201 may be bound to a labeling substance at a 3 'end, a 5' end, or a predetermined position.

In step (c), immunoassay may be performed using the first probe 300 and the second probe 400 that specifically bind to the nucleic acid sequence amplified by the loop-mediated isothermal amplification reaction have. 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 capable of hybridizing to a target nucleic acid molecule in a sample. Further, the immunoassay can be achieved by using an antibody specific to a hybrid substance formed by hybridization of the amplified nucleic acid sequence and the probe.

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, the step (c) may include reacting the result of the loop-mediated isothermal amplification reaction with the first probe 300; Diffusing a second probe (400) and an anti-first labeling material antibody (107) specifically binding the reactant to the second labeling substance (302) into a sequentially fixed test membrane; And determining the presence or absence of the microorganism through the color reaction.

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.

1 and 2, after the loop-mediated isothermal amplification reaction is reacted with the first probe 300 in step (c), the reactant is reacted with the second probe 400 and the anti- 1 labeling substance antibody 107 can be diffused in a sequentially fixed test membrane 103. The amplified DNA is immobilized on the reaction membrane 103 and confirmed by the second probe 400 .

The probe may bind to or hybridize with the DNA amplified by the loop-mediated isothermal amplification reaction to indicate presence or absence of the target microorganism. The probe is a substance specifically binding to the amplified DNA, and the kind thereof is not particularly limited as long as it can display the presence or absence of the DNA by specific interaction.

In one embodiment, in step (c), the first probe 300 may include an oligonucleotide that specifically binds to the gene of the target microorganism, and a second label material 302.

The first probe 300 specifically hybridizes with the gene because it contains an oligonucleotide that specifically binds to the gene of the target microorganism and the second probe 300 is hybridized specifically with the gene, Lt; / RTI >

That is, the second probe 400 may include a reaction material 401 and chromogenic particles 402 that specifically bind to the second labeling material 302. The second probe 400 can specifically bind to the second labeling material 302 when the first probe 300 is hybridized with the amplified DNA, It is possible to easily display whether the first probe 300 is coupled or not.

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 second probe 400 may include particles that can be colored by binding or aggregation so that the second probe 400 can be visually recognized. If the second probe 400 is visually confirmed by coloring, The presence or absence of the microorganism can be easily identified.

In the meantime, after the step (b), the antisense oligomer of the primer 201 or the S1 nucleic acid hydrolase (S1 nuclease) is reacted with the loop-mediated isothermal amplification reaction product before the step (c) .

That is, in the microorganism detection method, the specific gene of the target microorganism can be amplified using the primer 201, and the amplification can be quickly confirmed by immunoassay.

The microorganism detection method can detect the presence of microorganisms by detecting the amplified DNA. However, since the primer 201 and the first probe 300, which are not hybridized to a specific gene after the loop-mediated isothermal amplification reaction, If combined, it can lead to false positives.

FIG. 3 is a diagram showing a dimer formed by the interaction between the primer 201 and the first probe 300.

Referring to FIG. 3, the microorganism detection method can identify amplification of DNA through the labeling substance bound to the primer 201 of the amplified DNA. However, in the loop-mediated isothermal amplification reaction, The primer 201 and the first probe 300 can form a dimer due to interactions between molecules such as hydrogen bonding.

That is, since the duplex formed between the primer 201 and the first probe 300 includes both the first labeling substance 202 and the second labeling substance 302, the anti-first May be associated with the labeling material antibody 107 and identified by the second probe 400. In this case, even if the DNA of the microorganism is not amplified due to the absence of the target microorganism in the sample, a positive reaction may be induced, thereby causing a detection error.

Therefore, when the antisense oligomer of the primer 201 is reacted after the loop-mediated isothermal amplification reaction, the primer 201 forms a dimer with the antisense oligomer. Therefore, the primer 201 and the first probe 300) can not be formed and the false positives can be minimized.

When the S1 nucleic acid is reacted after the loop-mediated isothermal amplification reaction is completed, the unhybridized primer 201 present in a single strand is decomposed to form the first probe 300, And therefore the false positives can be excluded.

Microbial detection kit

A microbial detection kit according to another aspect of the present invention comprises a first solution comprising a primer 201 hybridized specifically to a gene of a target microorganism and to which a labeling substance is bound; A second solution comprising a first probe (300) specifically hybridized to the gene of the target microorganism; A sample pad 101 for dropping a sample of a microorganism, a conjugation pad 102 including a second probe 400, and a reaction membrane 102 having a test line 104 formed thereon. and a test membrane (103) arranged in this order.

The first solution may include a primer 201 hybridized specifically to a gene of a target microorganism and to which a first labeling substance 202 is bound.

The diagnostic strip 100 includes an anti-first sample pad 101 for loading the microbial sample, a conjugation pad 102 including the second probe 400, and a test line 102 for testing the test strip. A reaction membrane (test membrane) 103 having a membrane 104 formed thereon may be sequentially arranged.

The anti-first sample pad 101 may serve as an inlet through which the sample is directly injected. The solid-phase component contained in the sample may be filtered by using a porous material, 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 conjugation pad 102 may have a second probe 400 that specifically binds to the nucleic acid sequence amplified by the loop-mediated isothermal amplification reaction, and the second probe 400 may be condensed, A reactive substance 401 and chromogenic particles 402 that specifically bind to the second labeling substance 302.

The condensation pad 102 may be located downstream of the sample pad 101. If the microorganism sample reacted with the first probe 300 contains a large amount of specific DNA by the loop-mediated isothermal amplification reaction, the amplified DNA may be hybridized with the first probe 300. Therefore, when the microorganism sample is loaded on the sample pad 101 and then moved via the condensation pad 102, the amplified DNA is specific to the second probe 400 condensed on the condensation pad 102 Can be combined. That is, when the microorganism sample contains a large amount of characteristic DNA of the target microorganism by the loop-mediated isothermal amplification reaction, the second probe 400 condensed on the condensation pad 102 is separated from the first probe 300 Can be bound to the characteristic DNA by specifically binding with the second labeling substance (302).

The test membrane 103 may include the immobilized anti-first labeling 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 membrane 103 and the inspection line 104 may include the immobilized anti-first indicator material antibody 107.

That is, the DNA in the microorganism sample coupled with the second probe 400 via the condensation pad 102 may be bonded to the inspection line 104 located downstream, and may be identified by the naked eye or other means.

In one embodiment, when the second labeling material 302 of the first probe 300 is biotin, the condensation pad 102 is streptavidin that specifically binds to the biotin, And a second probe 400 in the form of a combination of gold particles. Since the second probe 400 can be coupled to the first probe 300 hybridized at a predetermined position of the amplified DNA, the second probe 400 can be visually confirmed at the inspection line 104 positioned at the downstream end.

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 an antisense oligomer of the primer 201, or a third solution containing S1 nucleic acid hydrolase (S1 nuclease).

The third solution may be added to the reaction product after the loop-mediated isothermal amplification reaction and reacted for a predetermined period of time. By this reaction, the dimer formation between the primer 201 and the first probe 300 is inhibited, The false positives that can be induced 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.

Preparation Example 1: Preparation of a primer and a first probe to which a labeling substance was bound

A primer and a first probe hybridizing to a gene characteristic to a specific microorganism were prepared as shown in Table 1 below. DIG means digoxigenin, and BTN means biotin. The primer was labeled with digoxigenin at the 5 'end as the first labeling substance, and biotin was attached at the 5' end as the second labeling substance to the first probe.

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

Production Example 2: Fabrication of second probe

Streptavidin (streptoavidin) and gold particles were combined to prepare a second probe. To prepare the second probe, streptavidin was diluted in a 0.5 × PBS solution (phosphate buffered saline, PBS) at a concentration of mg / ml. 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 (second probe) was refrigerated 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 of the nitrocellulose pad, dried at 37 ° C in a thermostat, .

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: Loop-mediated Isothermal Amplification (LAMP)

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 preculture solution through a filter. The liquid component was allowed to stand at room temperature for a predetermined time, and then the loop-mediated isothermal amplification reaction was performed using the primer sequence of Preparation Example 1.

40 pmole of the forward primer, reverse primer, FIP and BIP primer of Preparation Example 1, 5 pmole of 10 mM dNTPs, 8 U Bst DNA polymerase large fragment (New England Biolabs, USA), 1 X ThermoPol reaction buffer (20 mM Tris-HCl, 10 mM KCL, NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100) and 2 μl of the separated liquid components were adjusted to a total volume of 50 μl to prepare an isothermal amplification reaction solution. The reaction mixture was denatured at 95 ° C for 10 minutes, subjected to DNA synthesis reaction at 55 to 65 ° C for 60 minutes and then incubated at 80 ° C for 10 minutes to inactivate Bst DNA polymerase, and the reaction was terminated.

After completion of the reaction, 40 pmole of the first probe of Preparation Example 1 was added and reacted for a predetermined time.

Experimental Example 2: Evaluation using a lateral flow diagnostic strip

The result of the loop-mediated isothermal amplification reaction of Experimental Example 1 was added dropwise to the diagnostic strip of Preparation Example 3 and developed. The results of the loop-mediated isothermal amplification reaction showed a color reaction in the test line, but no chromogenic 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-first marker substance antibody
108: Second antibody
201: Primer
202: First label substance
300: first probe
302: Second labeling substance
400: second probe
401: Reactive material
402: coloring particles

Claims (14)

(a) preparing a primer hybridized specifically to a gene of a target microorganism and to which a first labeling substance is bound;
(b) performing a Loop-mediated Isothermal Amplification (LAMP) using the primer; And
(c) performing immunoassay using a first probe and a second probe that specifically bind to the nucleic acid sequence amplified by the loop-mediated isothermal amplification reaction. The method according to claim 1,
In the step (a), a labeling substance is bound to the 3 'end, the 5' end or a predetermined position of the primer. 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 first probe comprises an oligonucleotide that specifically binds to a gene of the target microorganism, and a second marker substance. 5. The method of claim 4,
Wherein the step (c) comprises: reacting the loop-mediated isothermal amplification reaction product with the first probe;
Diffusing the reactant into a reaction membrane in which a second probe and an anti-first labeling substance antibody specifically binding to the second labeling substance are sequentially immobilized; And
And determining the presence or absence of the microorganism through a color reaction. 6. The method of claim 5,
Wherein the second probe comprises a reactive substance and a coloring particle that specifically bind to the second labeling substance. The method according to claim 6,
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,
Reacting the resultant loop-mediated isothermal amplification reaction product with the antisense oligomer of the primer or the S1 nucleic acid hydrolase (S1 nuclease) before the step (b) after the step (b) Way. A first solution comprising a primer specifically hybridized to a gene of a target microorganism and to which a labeling substance is bound;
A second solution comprising a first probe that specifically hybridizes to the gene of the target microorganism; And
A diagnostic strip in which a sample pad for dropping a microorganism sample, a conjugation pad including a second probe, and a test membrane on which a test line is formed are sequentially arranged A microorganism detection kit. 10. The method of claim 9,
Further comprising a third solution comprising an antisense oligomer of said primer, or S1 nucleic acid hydrolase (S1 nuclease). 10. The method of claim 9,
Wherein the second probe comprises a reactive substance and a coloring particle that specifically bind to the second labeling substance. 12. The method of claim 11,
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. 10. The method of claim 9,
Wherein the test line is an immobilized microorganism detection kit in which the anti-first labeling substance antibody is immobilized. 10. The method of claim 9,
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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