KR20210097242A - Primer set for high sensitive multiplex loop-mediated isothermal amplification reaction for detection and identification of Mycobacterium tuberculosis and Nontuberculous mycobacteria - Google Patents

Abstract

The present invention relates to a primer set for a multiplex loop-mediated isothermal amplification reaction capable of simultaneously identifying and detecting Mycobacterium tuberculosis (MTB) and Nontuberculous mycobacteria (NTM). According to the present invention, MTB and NTM can be detected rapidly and accurately without heavy and complex PCR equipment and can be identified and diagnosed. Therefore, the present invention is expected to be used as a molecular diagnostic test to replace conventional detection methods for tuberculosis.

Description

Primer set for high sensitive multiplex loop-mediated isothermal amplification reaction for detection and identification of Mycobacterium tuberculosis and Nontuberculous mycobacteria

The present invention relates to a primer set for multiple isothermal amplification reactions capable of simultaneously discriminating and detecting Mycobacterium tuberculosis (MTB) and Nontuberculous mycobacteria (NTM).

Tuberculosis is a droplet-infected disease caused by Mycobacterium tuberculosis (MTB) and is one of the leading causes of infectious disease deaths that have killed millions of people over the past centuries. It is also estimated to cause 10 million new cases and approximately 2 million deaths each year.

On the other hand, Nontuberculous mycobacteria (NTM) are bacteria that are widely present in natural environments such as water and soil. Epidemiologically, M, avium complex accounts for 60-80%, in foreign countries, M. kansasii accounts for 15-20%, and in Korea, M. abscessus accounts for 20-30%, and NTM is isolated from clinical specimens in Korea. The number of patients diagnosed and treated for NTM lung disease is increasing significantly.

Existing methods for detecting tuberculosis include chest radiography, acid fast staining of direct smears, and culturing in solid and liquid media. Confirming positivity in culture is a confirmatory method, but there is a limitation in that the culture period is very long (6-8 weeks). In addition, in the case of acid-resistant staining of direct smear samples, there is a disadvantage that MTB and NTM cannot be distinguished. Therefore, recently, Mycobacterium tuberculosis polymerase chain reaction (PCR) test is used for the sensitive and rapid detection and identification of MTB and NTM by supplementing the shortcomings of these smear and culture tests for the classification and diagnosis of MTB and NTM. The tuberculosis guideline also recommends using PCR for identification of suspected patients. However, MTB and NTM detection using PCR requires skilled inspectors and an advanced laboratory base. Therefore, there are limitations in testing in areas where specialized medical services are not available or where it is necessary to perform a large number of screening tests in a short period of time.

Recently, a loop-mediated isothermal amplification reaction (LAMP) that amplifies a gene at a constant temperature without temperature change has been developed. The LAMP method uses six primers complementary to the template to recognize eight different sites and proceed with gene amplification. Compared with the existing PCR method, the test time can be shortened in that it can be amplified without a change in temperature, and the test can be performed without expensive equipment for temperature change.

Accordingly, the present inventors completed the present invention by intensively studying a method for simultaneously identifying MTB and NTM using the LAMP method without an experienced tester and expensive equipment.

Norihiro, T. et. al. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature protocols. 2008;3:5.

The technical problem to be achieved by the present invention is to provide a Mycobacterium tuberculosis detection primer set and a Mycobacterium tuberculosis and Mycobacterium tuberculosis detection primer set, and a primer set capable of discriminating and detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis, including each of the primer sets, and To provide a kit and a method for differentially detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis using the same.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a LAMP (Loop-mediated isothermal amplification) primer set for detecting Mycobacterium tuberculosis (MTB) comprising the primers of SEQ ID NOs: 1 to 6.

As an embodiment of the present invention, the LAMP primer set for detecting Mycobacterium tuberculosis may include an MTB probe, and the MTB probe may include the nucleotide sequence of SEQ ID NO: 7.

In addition, the present invention provides a LAMP primer set for detecting Mycobacterium tuberculosis and Non-tuberculosis bacterium (NTM), comprising the primers of SEQ ID NOs: 8 to 13.

In one embodiment of the present invention, the LAMP primer set for detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis may include an NTM probe, and the NTM probe may include the nucleotide sequence of SEQ ID NO: 14.

In addition, the present invention provides a LAMP primer kit for differential detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis comprising the MTB primer set of SEQ ID NOs: 1 to 6 and the NTM primer set of SEQ ID NOs: 8 to 13.

In addition, the present invention provides a LAMP kit for diagnosing tuberculosis comprising the MTB primer set of SEQ ID NOs: 1 to 6 and the NTM primer set of SEQ ID NOs: 8 to 13.

In one embodiment of the present invention, the kit may further include an internal control (IC) primer set of SEQ ID NOs: 15 to 20.

As another embodiment of the present invention, the MTB primer set included in the kit may further include an MTB probe comprising the nucleotide sequence of SEQ ID NO: 7, and the NTM primer set includes the nucleotide sequence of SEQ ID NO: 14 An NTM probe including a nucleotide sequence may be further included, and the IC primer set may further include an IC probe including the nucleotide sequence of SEQ ID NO: 21.

In another embodiment of the present invention, the MTB probe, the NTM probe and the IC probe are respectively FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (2',4',5) ',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine ( tetramethyl rhodamine), FITC (fluorescein isothiocyanate), oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX ( 6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and CYADICA One or more fluorescent substances selected from the group consisting of lebocyanine (thiadicarbocyanine) may be attached thereto, and each of the probes may have fluorescent materials emitting different wavelengths attached thereto.

In another embodiment of the present invention, the kit may further include a reagent for performing an amplification reaction, and the reagent may include one or more selected from the group consisting of DNA polymerase, dNTPs, and a buffer.

In addition, the present invention comprises the steps of (1) isolating genomic DNA (gDNA) from a biological sample; (2) MTB primer set of SEQ ID NOs: 1 to 6 using the isolated gDNA as a template; and amplifying the target sequence by performing an isothermal amplification reaction using the MTB and NTM differential diagnostic composition comprising the MTB and NTM primer sets of SEQ ID NOs: 8 to 13; and (3) detecting the amplified product; provides a differential detection method comprising Mycobacterium tuberculosis and Mycobacterium tuberculosis.

In addition, the present invention comprises the steps of (1) isolating genomic DNA (gDNA) from a biological sample; (2) MTB primer set of SEQ ID NOs: 1 to 6 using the isolated gDNA as a template; NTM primer set of SEQ ID NOs: 8 to 13; and performing an isothermal amplification reaction using a kit comprising the IC primer set of SEQ ID NOs: 15 to 20 to amplify the target sequence; and (3) detecting the amplified product; provides an information providing method for diagnosing tuberculosis, including.

In one embodiment of the present invention, the biological sample may be at least one selected from the group consisting of sputum, bronchial aspiration and bronchial washing, and body fluid.

As another embodiment of the present invention, step (3) may be performed by one or more methods selected from the group consisting of DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, and phosphorescence measurement, but preferably fluorescence measurement can depend on the method.

As another embodiment of the present invention, the method comprises: MTB primer set after step (3); and when all of the products amplified by the NTM primer set are detected, it is interpreted as MTB positive. may additionally include.

As another embodiment of the present invention, the method may include the step of interpreting as negative when the amplification product by the internal control primer set is detected and the amplification product by the other primer set is not detected after step (3) there is.

As another embodiment of the present invention, the method may further include quantifying MTB and/or NTM by measuring the detection time of step (3) after step (3). The "quantification" may be to quantify the fluorescent material detection time according to the DNA concentration compared to a standard curve when performing an amplification reaction using the primer set according to the present invention, and preferably, the amplification product of step (c) is fluoresced. By detecting using an indicator, the detection time of the fluorescent substance may be measured to quantify the DNA concentration.

The primer set of the present invention is based on real-time multiplex LAMP, and shortens the identification time of the causative agent of lung disease compared to conventional PCR and real-time PCR-based detection, and is based on LAMP. There is an advantage in that a simple, economical and lightweight device can be used compared to the conventional PCR device because it does not require a temperature change. In addition, the present invention is advantageous in terms of time and cost in that both MTB and NTM can be detected and discriminated at the same time by multiplex testing, so it is expected to be used as a molecular diagnostic test method replacing the existing tuberculosis detection method.

1 is a view showing the position of the LAMP primer set in the IS6110 gene sequence, which is a target gene of MTB.
2 is a view showing the position of the LAMP primer set in the RpoB gene sequence, which is a target gene of NTM.
3 is a view showing the detection test results of the multiplex LAMP kit (Cy5 (MTB), TexasRED (NTM), HEX (GAPDH)) of the present invention.

The present inventors have completed the present invention by intensively researching a method for quickly and accurately identifying pathogens of infectious lung disease patients in the field.

Specifically, the present inventors have found that Mycobacterium tuberculosis was prepared primers specific set the RpoB of:: (NTM Nontuberculous mycobacteria) with LAMP-based (Mycobacterium tuberculosis MTB) and Mycobacterium tuberculosis specific primers and non-tuberculosis mycobacteria in IS6110 gene of. Unlike conventional PCR, the LAMP-based primer set of the present invention does not require a temperature change, so a simple and light amplification device can be used, and the time required for analysis can be dramatically reduced.

In addition, the present invention can shorten the cell identification time by enabling the detection and differentiation of mycobacteria and non-mycobacteria at the same time by performing a multiplex LAMP including primers specific set to a specific primer set and the RpoB the IS6110.

In addition, the present invention can improve the reliability of the multiplex LAMP analysis result by preparing a primer set specific for the GAPDH gene and providing it as an internal control primer set.

Specifically, the present invention provides a primer set that specifically acts on MTB (MTB primer set), a primer set that simultaneously acts on 25 types of NTM and MTB (NTM primer set), and a primer capable of confirming whether amplification is performed with an internal control A set (IC primer set) is provided.

In addition, the present invention provides a composition for detecting MTB bacteria comprising the primer set. SEQ ID NOs: 1-6 in Table 1 below are six primer sets for MTB (MTB primers), SEQ ID NOs: 8-13 are 25 kinds of NTM and six primer sets for MTB (NTM primers), and SEQ ID NO: 15 -20 is a set of 6 primers (Internal control primer) that can amplify the GAPDH gene as an internal control. The primer set of Table 1 and the composition including the same can be used for the detection of MTB and NTM bacteria using the Multiplex LAMP method.

The composition of the multiplex LAMP primer set for differential detection of MTB and NTM of the present invention is shown in Table 3 below, and the reaction time and temperature are shown in Table 4 below.

The present invention can provide an LAPM kit for diagnosing tuberculosis including the three primer sets, and the LAMP kit is used to perform multiplex LAMP to obtain a cell identification result in one test. Specifically, when amplified by both the MTB primer and the NTM primer, it is interpreted as MTB. If the NTM primer is amplified but the MTB primer is not amplified, it is interpreted as NTM. When the test result is negative, it is interpreted as true negative if amplification is done by the internal control primer. If the result is negative or the internal control primer is also negative, it is recommended to retest.

Meanwhile, the three primer sets of the present invention may each independently include a probe, and each probe may have a fluorescent material having a different emission wavelength range attached thereto.

In the present invention, cell identification results can be quickly obtained in one test by performing multiplex real-time LAMP using the three primer sets including each probe emitting different fluorescence.

The fluorescent material attached to each probe is not limited as long as it emits fluorescence, but non-limiting examples thereof include FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein, HEX (2', 4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetra methyl rhodamine, FITC (fluorescein isothiocyanate), oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein) ), ROX(6-Carboxyl-XRhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl)ethylenediamine), cyanine series dyes and thiadicarbocyanine.

In addition, the present invention isolates genomic DNA (gDNA) from a biological sample of an individual in need of tuberculosis diagnosis, amplifies a target sequence by performing LAMP using the kit using the gDNA as a template, and detects the amplification product. It is possible to provide a method for diagnosing tuberculosis comprising the steps of:

In the present invention, the "individual" is not limited as long as it is a mammal requiring a diagnosis of tuberculosis, but preferably a human. In addition, the "biological sample" in the present invention may include cells or tissues of various parts of the body, including sputum, bronchial aspiration and bronchial washing, body fluid, etc. there is.

In the present invention, "LAMP; Loop-mediated isothermal amplification" is a method of performing a reaction under isothermal conditions using polymerases such as Bst and Gsp that do not require a PCR cycle unlike the existing PCR method. As a method of verifying the presence or absence of amplification by synthesizing both ends into a loop structure using four primers as , Preferably, a reagent for detecting fluorescence may be used, but the present invention is not limited thereto.

In addition, in the present invention, the "multiplex LAMP" can simultaneously detect various genes, and preferably, simultaneously detect MTB and/or NTM in a sample to determine the presence or absence of MTB and/or NTM in the sample. It can be determined, but is not limited thereto.

In addition, in the present invention, “multiplex real-time LAMP” is a method for checking in real time whether or not the various genes are amplified. It is possible to determine the presence or absence of MTB and/or NTM in real time.

The kit comprising the primer set of the present invention is preferably used for isothermal amplification (LAMP) for the diagnosis of tuberculosis, and the isothermal amplification reaction is a single-step amplification of target DNA by LAMP It may be a (one-step) LAMP, may be a real-time LAMP (real-time LAMP), or may be a combination thereof.

In the present invention, the term "primer" means a template under suitable conditions (for example, four different nucleoside triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature. - refers to a single-stranded oligonucleotide that can serve as a starting point for directing DNA synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template.

The primer of the present invention can be chemically synthesized using methods known in the art, such as, for example, phosphoramidite solid support method. In addition, it can be modified by methylation, encapsulation, etc. by a known method. In addition, if necessary, the primer of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horseradish peroxidase, alkaline phosphatase), radioactive isotopes (eg, 32P), fluorescent molecules, chemical groups (eg, biotin), and the like. there is.

In the present invention, "inner primer" refers to a single-stranded oligonucleotide capable of binding to a template DNA and serving as a starting point for synthesis of a new DNA chain.

In the present invention, "outer primer" refers to a single-stranded oligonucleotide that binds to the template DNA outside the position where the inner primer binds to the template DNA. After the chain is elongated, a strand displacement occurs due to the binding of the external primer and the template DNA, and the previously formed chain is separated.

In the present invention, the “loop primer” refers to an initial stem loop structure formed by binding the inner primer and the outer primer to the template DNA by increasing the number of loop structures as a result. It refers to a single-stranded oligonucleotide that can act as a starting point for nucleotide synthesis, allowing the reaction to be accelerated.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Materials and Methods]

1. DNA of MTB Bacteria and NTM

Nucleic acids (DNA) of MTB bacteria and NTM extracted from the sputum and bronchial aspirates of tuberculosis patients and non-tuberculous mycobacterium-infected patients were obtained at Korea University Guro Hospital, and the infection of each sample was confirmed by real-time PCR at the hospital. became

2. Clinical specimens

A total of 90 samples, 30 MTB, 30 NTM, and 30 tuberculosis-negative, were obtained from Korea University Guro Hospital and used.

3. Primer set for MTB and NTM detection, LAMP mixture composition and reaction conditions

Based on the rpoB gene sequence of IS6110 gene of MTB bacteria sequence and Mycobacterium bacteria (both MTB, NTM) was constructed by designing MTB bacteria, and 25 kinds of primer / probe set for NTM detected (see Figs. 1 and 2). A GAPDH gene amplification primer/probe set was prepared as an internal control. Specific information of each primer/probe set is summarized in Table 1 below. The composition of the real-time LAMP primer mix is shown in Table 2 below, the reagent (using M-monitor LAMP kit) composition of the reaction mixture for real-time LAMP is shown in Table 3 below, and the real-time LAMP is shown in Table 4 below. was carried out under the reaction conditions of

SEQ ID NO: Name Sequence (5'-3') length MTB primer/probe set One IS6110_F3 GGT CGG AAG CTC CTA TGA CA 20 2 IS6110_B3 TAG GCA GCC TCG AGT TCG 18 3 IS6110_FIP AGG GCT TGC CGG GTT TGA TCA TGC ACT AGC CGA GAC GA 38 4 IS6110_BIP CGG TCC ATC GAG GAT GTC GAG TCG CCG CAG TAC TGG TAG A 40 5 IS6110_FLP CTC GGT CTT GTA TAG GCC GT 20 6 IS6110_BLP1 ACC GCG CGC TGG GTC GA 17 7 IS6110_BLP1_PROBE2_Cy5 GTC AGT GCA GGC TCC CGT GTT AGG ACG AGG GTA GGA CCG CGC GCT GGG TCG A 52 NTM primer/probe set 8 RpoB NTM F3 CCG GTC ACC GTG CTG 15 9 RpoB NTM B3 GTA RCG CTT CTC CTT GAA GAA C 22 10 RpoB NTM FIP TGT TGT CCT TCT CCA GIG TII GCT GGA CCA ICG AGC A 37 11 RpoB NTM BIP TGG ACA TCT ACC GCA AGC TGC GTT YTC CAR CAG GGT CTG C 40 12 RpoB NTM FLP-2 CGG AGA AIC CGA AIC GCT C 17 13 RpoB NTM BLP2 CCG C CG ACC AAA GAG TCA GC 20 14 RPOB NTM- BLP2_TEX TEXASRED-CCG CCG ACC AAA GAG TCA GC-BHQ1 20 IC primer/probe set 15 GAPDH_DNA_LAMP_F3 TCC GCA CAT CCC GGT ATG 18 16 GAPDH_DNA_LAMP_B3 TCT GCA GAT AGG AAG GGC TT 20 17 GAPDH_DNA_LAMP_FIP TGG GGG CAT TGA AGG GGT GAT GAG GGT TCT TTG TGC TGA G 40 18 GAPDH_DNA_LAMP_BIP CTG TGG CAT CCC TGG GAC TGG TGA GGA ACT GGG AGA TCC A 40 19 GAPDH_DNA_LAMP_LF ATG GGC CTT TCT CCC CTG C 19 20 GAPDH_DNA_LAMP_LB GGG GAA GGT TGA GCC TTT ACT 21 21 GAPDH_DNA_LAMP_LB_Probe4 HEX CGG GCC CGT ACA AAG GGA ACA CCC ACA CTC CGG GGG AAG GTT GAG CCT TTA CT 53 22 lamp_PROBE2_QUANCHER CCT ACC CTC GTC CTA ACA CGG GAG CCT GCA CTG AC-BHQ2 35 23 lamp_PROBE4_QUANCHER GAG TGT GGG TGT TCC CTT TGT ACG GGC CCG-BHQ1 30

SFTS LAMP primer mix composition Name uM IS6110_F3 4 IS6110_B3 4 IS6110_FIP 32 IS6110_BIP 32 IS6110_FLP 10 IS6110_BLP1 4 IS6110_BLP1_PROBE2_Cy5 6 RpoB NTM F3 2 RpoB NTM B3 2 poB NTM FIP 18 poB NTM BIP 18 poB NTM FLP-2 18 RpoB NTM BLP2 18 RPOB NTM- BLP2_TEX 18 Composition of Internal Control LAMP primer mix Name uM GAPDH_DNA_LAMP_F3 4 GAPDH_DNA_LAMP_B3 4 GAPDH_DNA_LAMP_FIP 32 GAPDH_DNA_LAMP_BIP 32 GAPDH_DNA_LAMP_LF 10 GAPDH_DNA_LAMP_LB 4 GAPDH_DNA_LAMP_LB_Probe4 HEX 6

division volume RM 12.5 EM 2 Distilled water 3.625 Primer mix 0.5 (IS6110 MTB) 1.5 (RpoB NTM) 0.5 (GAPDH Internal control) QUANCHER P4 2.5 Template 2.5 TOTAL 25

One-step RT-LAMP reaction temperature and time conditions temperature reaction time 65 ℃ 1 min 60 Cycles 80 ℃ 5 minutes total reaction time 65 minutes

[Experiment result]

Example 1. Detection test of LAMP primer/probe set

Using the MTB, NTM, and IC primer/probe set labeled with different fluorescence (Cy5, Texas Red, Hex) for actual MTB-positive samples, MTB and NTM-positive samples, and negative samples, distilled water (DW) Real-time LAMP was performed.

As a result, as shown in FIG. 3, it was confirmed that the primer/probe set for LAMP of the present invention can simultaneously detect MTB, NTM, and GAPDH genes.

Example 2. Identification of cross-reactivity to NTM of LAMP primer/probe set

Making the plasmid DNA inserted into the IS6110 gene of MTB, and the plasmid DNA was diluted in deionized water to 10 to ¹ was 1X10 determine the sensitivity of ^{7-10 1} concentrations MTB primer / probe set. Nucleic acids used for testing were stored at -50°C when not in use. As a result of verifying the analytical ability by concentration using a dilution factor of 1/10 ^{, amplification was confirmed in 10 5} copies (approximately 5 x 102 cells). . Table 4 below shows the Ct values of the Mycobacterium tuberculosis LAMP primer set according to the dilution concentration.

sample IS6110 (Cy5) CT RFU IS6110　 Plasmid　 10^7 13.7 1479 IS6110　 Plasmid　 10^6 16.31 1624 IS6110　 Plasmid　 10^5 20.88 1602 IS6110　 Plasmid　 10^4 N/A -1.18 IS6110　 Plasmid　 10^3 N/A -1.2 IS6110　 Plasmid　 10^2 N/A 0.643 IS6110　 Plasmid　 10^1 N/A -1.12 DW N/A 1.68

Example 3. Confirmation of detection Ct values in MTB, NTM, and tuberculosis-negative clinical samples of LAMP primer/probe set

Clinical trials were conducted with a total of 90 samples, 30 MTB, 30 NTM, and 30 tuberculosis-negative, provided by Korea University Guro Hospital. Real-time LAMP was performed on the DNA sample extracted from the specimen, and the detection result of Mycobacterium tuberculosis bacteria was confirmed.

The performance evaluation results of the tuberculosis Multiplex LAMP kit for 30 MTB-positive samples are shown in Table 6 below, and the performance evaluation results of the tuberculosis Multiplex LAMP kit for 30 NTM-positive samples are shown in Table 7 below. The performance evaluation results of the tuberculosis Multiplex LAMP kit for 30 specimens are shown in Table 8 below.

Samples IS6110 (Cy5) RPOBN™ (Texas Red) GAPDH (Hex) Ct RFU Ct RFU Ct RFU MTB-Positive 01 21 1646 26.99 1677 31.37 1595 MTB-Positive 05 22.02 1277 35.98 1207 29.93 1993 MTB-Positive 06 18.42 1954 23.89 6200 26.34 447 MTB-Positive 08 20.07 1382 26.33 3971 23.7 2004 MTB-Positive 12 22.57 1396 34.06 3037 26.62 2279 MTB-Positive 13 22.07 1354 34.05 1392 26.42 2277 MTB-Positive 14 20.02 1384 22.9 5005 26.55 875 MTB-Positive 15 28.7 949 40.48 1382 27.26 1855 MTB-Positive 17 19.67 1785 21.83 6588 26.48 182 MTB-Positive 18 18.02 1514 30.2 4514 N/A 56.6 MTB-Positive 19 24.33 1181 26.11 6148 28.79 136 MTB-Positive 21 21.08 1817 26.65 1908 30.92 1708 MTB-Positive 24 27.28 1129 40.5 1513 28.33 2111 MTB-Positive 25 20.22 1882 26.82 1650 32.13 457 MTB-Positive 27 27.12 1068 38.98 1478 26.44 2032 MTB-Positive 29 20.43 1817 29.94 6378 28.16 1625 MTB-Positive 30 20.44 1228 34.74 1203 31.44 1712 MTB-Positive 31 21.24 1435 36.22 1456 31.48 2015 MTB-Positive 32 20.14 1277 33.79 1296 26.73 2091 MTB-Positive 33 22.19 1232 36.95 1289 28.55 2041 MTB-Positive 34 21 1317 27.55 4130 27.89 170 MTB-Positive 36 18.35 1710 25.77 5231 45.61 173 MTB-Positive 38 20.32 1558 32.39 1324 40.86 1694 MTB-Positive 38 24.18 1136 36.22 1453 23.99 2264 MTB-Positive 39 22.77 1567 36.34 1288 38.83 1862 MTB-Positive 40 25.78 1620 38.43 1294 38.9 1932 MTB-Positive 49 33.11 2183 39.55 1667 25.29 2802 MTB-Positive 54 19.28 1977 27.68 6246 35.51 1055 MTB-Positive 58 20.56 1984 29.51 1759 28.73 2219 MTB-Positive 60 18.48 2137 27.07 1863 29.21 2314

Samples IS6110 (Cy5) RPOBN™ (Texas Red) GAPDH (Hex) Ct RFU Ct RFU Ct RFU NTM-Positive 006 N/A 95.5 19.6 3675 N/A 75.1 NTM-Positive 017 N/A 71.2 19.27 2907 N/A 62.4 NTM-Positive 074 N/A 109 23.3 2809 27.62 1058 NTM-Positive077 N/A 75.8 21.78 3778 29.21 159 NTM-Positive 079 N/A 131 22.27 4871 28.16 170 NTM-Positive 080 N/A 105 22.15 3316 27.02 176 NTM-Positive 081 N/A 142 19.96 4821 26.22 171 NTM-Positive 086 N/A 128 19.39 3788 25.23 130 NTM-Positive088 N/A 98.2 24.66 4039 30.9 141 NTM-Positive 089 N/A 77.7 21.89 2973 27.18 92.1 NTM-Positive 090 N/A 76.9 20.12 3579 25.87 116 NTM-Positive 091 N/A 85.6 28.89 3558 31.31 195 NTM-Positive 132 N/A 92.6 25.42 3496 30.63 143 NTM-Positive 134 N/A 125 22.71 3707 27.99 164 NTM-Positive 135 N/A 103 24.17 3927 30.26 144 NTM-Positive 136 N/A 94.6 21.04 3680 24.15 169 NTM-Positive 137 N/A 119 22.32 4125 27.76 157 NTM-Positive 138 N/A 38.8 21.67 3138 28.53 133 NTM-Positive 139 N/A 77.1 20.16 3288 26.97 135 NTM-Positive 140 N/A 99.7 23.23 4042 30.56 128 NTM-Positive 141 N/A 91.1 24.57 3768 31.68 207 NTM-Positive 142 N/A 90.1 22.7 3570 29.14 140 NTM-Positive143 N/A 74.2 28.93 3573 29.44 686 NTM-Positive 144 N/A 94.2 18.76 3641 N/A 73.2 NTM-Positive 145 N/A 111 18.13 3224 N/A 74 NTM-Positive 146 N/A 77.7 23.26 2997 51.33 170 NTM-Positive 152 N/A 129 19.45 3992 20.01 204 NTM-Positive 153 N/A 91.8 27.81 4871 30.02 415 NTM-Positive 154 N/A 105 26.23 4753 29.36 559 NTM-Positive 155 N/A 80.4 28.02 3940 29.63 455

Samples IS6110 (Cy5) RPOBN™ (Texas Red) GAPDH (Hex) Ct RFU Ct RFU Ct RFU TB negative 090 N/A 0.99 N/A 51.7 35.14 2047 TB negative 091 N/A 3.44 N/A 53.3 33.4 2084 TB negative 092 N/A 3.86 N/A 63.4 34.14 2139 TB negative 093 N/A 2.36 N/A 92.5 38.14 1869 TB negative 094 N/A 0.824 N/A 72.2 32.31 2090 TB negative 095 N/A 2.13 N/A 45.6 31.49 2324 TB negative 096 N/A 3.61 N/A 383 30.17 2178 TB negative 097 N/A 3.96 N/A 82 28.49 2590 TB negative 098 N/A 1.6 N/A 93.2 27.47 2396 TB negative 100 N/A 6.54 N/A 187 25.99 2412 TB negative 101 N/A 2.79 N/A 558 26.19 2287 TB negative 102 N/A 0.751 N/A 51.3 29.51 2200 TB negative 103 N/A 1.73 N/A 553 29.06 2189 TB negative 104 N/A 3.61 N/A 123 24.98 2303 TB negative 105 N/A 1.81 N/A 105 38.61 2049 TB negative 106 N/A -0.087 N/A 37.1 30.23 2135 TB negative 107 N/A 2.25 N/A 60 29.91 2315 TB negative 108 N/A 2.2 N/A 43.2 29.55 2440 TB negative 109 N/A 1.53 N/A 42.7 29.47 2371 TB negative 110 N/A 2.09 N/A 51.7 29.8 2222 TB negative 111 N/A 2.95 N/A 68.4 28.16 2282 TB negative 112 N/A -0.0576 N/A 56.3 30.86 2482 TB negative 113 N/A 0.954 N/A 58.4 31.96 2330 TB negative 114 N/A 1.41 N/A 71.9 33.82 2239 TB negative 115 N/A 5.06 N/A 163 25.98 2437 TB negative 131 N/A 4.06 N/A 59.1 28.19 2201 TB negative 132 N/A -0.876 N/A 344 31.26 2605 TB negative 133 N/A 3.53 N/A 100 26.37 2364 TB negative 134 N/A 1.86 N/A 86.3 30.41 2108 TB negative 135 N/A 5.48 N/A 136 25.12 2106

As can be seen from the results in Tables 6 to 8, the primer/probe kit for the multiplex LAMP showed 100% identical results to the hospital results without non-specific reactions for all test samples.

Example 4. Confirmation of effect comparison with the conventional LAMP primer set

As a conventional LAMP primer set developed for tuberculosis diagnosis, one targeting the gyrB gene of MTB (KR 10-2017-0038995) and one targeting the IS6110 gene of MTB as an amplification target (KR 10-2017-0038995) ) is there. Accordingly, it was attempted to confirm whether the primer set of the present invention has a better effect on diagnosing tuberculosis than the conventional primer set. Specific information on the primer set of the present invention and the conventional LAMP primer set to be compared is shown in Table 9 below.

SEQ ID NO: Name Sequence (5'-3') length MTB (KR 10-2017-0038995) gyrB primer set 24 gyrB_F3 GTGAACAAGTACGCCAAG 18 25 gyrB_B3 CACAGCCTTGTTCACAAC 18 26 gyrB_FIP TGACCTTCACCGAGATCACAGCTACTGAAGGACAAGGA 38 27 gyrB_BIP GCCAGACCAAGACCAAGTTGTGTTCGTTACAGACCTTCT 39 28 gyrB_FLP CCTTCCCGGATATCGTCA 18 29 gyrB_BLP1 GCAACACCGAGGTCAAAT 18 MTB (KR 10-2017-0038995) IS6110 primer set 30 IS6110_F3 TTCGCAATTCGGCGTTGT 18 31 IS6110_B3 ATGTCAGGTGGTTCATCG 18 32 IS6110_FIP TCTACTTGGTGTTGGCTGCGGATTCTTCGGTCGTGGTC 38 33 IS6110_BIP GACCTCACTGATCGCTGCCGGATGGTCGCAGAGATCC 37 34 IS6110_FLP GAGACGGTGCGTAAGTGG 18 35 IS6110_BLP1 ACTCCGAATCGTGCTGAC 18

4-1. Check limit of detection (LOD)

To compare and confirm the tuberculosis detection sensitivity of the LAMP primer set of the present invention and the conventional LAMP primer set. Specifically, 1 ng/μL of MTB genomic DNA was diluted to 10 with tertiary distilled water to confirm the sensitivity of each primer set. Nucleic acids used for testing were stored at -50°C when not in use. Analytical performance for each concentration was confirmed using a 1/10 dilution factor. Table 10 below compares the results.

MTB-Positive 34 Multiplex MTB/NTM LAMP assay Existing patented LAMP primer set IS6110 (Cy5) RPOBNTM:BLP2CG(TEX) IC(HEX) GyrB IS6110 CT RFU CT RFU CT RFU Ct RFU Ct RFU One 21 1317 27.55 4130 27.89 170 28.2 6440 22.62 21559 10 ^-1 30.86 3956 48.33 1159 46.79 81.2 52.48 13201 26.8 15645 10 ^-2 32.06 4190 50.36 1009 46.84 86.3 N/A 6840 41.77 17078 10 ^-3 N/A 4.08 N/A 41.1 N/A 3.78 N/A 5906 N/A 6009 10 ^-4 N/A 4.77 N/A 52 N/A 4.29 N/A 5612 N/A 3729 10 ^-5 N/A 2.5 N/A 37.3 N/A 5.11 N/A 3797 N/A 4845 N.C (hg DNA) N/A 9.44 N/A 168 35.04 2236 N/A 6393 N/A 2724 D.W. N/A 0.967 N/A 30.9 N/A 0.384 N/A 8280 N/A 684

As a result, it was confirmed that the primer set of the present invention was able to detect MTB in a lower dilution sample than the conventional gyrB primer set, and exhibited a significantly faster Ct value than the conventional IS6110 primer set.

4-2. Confirmation of Ct values in MTB, NTM, and tuberculosis-negative clinical specimens

The performance tests of the primer set of the present invention and the conventional primer set were performed on a total of 30 samples, including 10 MTB, 10 NTM, and 10 tuberculosis-negative samples, provided by Korea University Guro Hospital. The results for the MTB-positive sample are summarized in Table 11, the results for the NTM-positive sample are shown in Table 12, and the results for the tuberculosis-negative sample are summarized in Table 13 below.

sample IS6110 (Cy5) RPOBNTM:BLP2CG(TEX) IC(HEX) Re-IS6110 Re-GyrB CT RFU CT RFU CT RFU Ct RFU Ct RFU TB-Positive 4 25.24 4573 30.56 2333 19.03 2377 25.62 7182 38.46 11475 TB-Positive 10 20.29 4476 30.46 2031 34.44 1662 29.29 20662 42.72 15880 TB-Positive 35 23.33 4540 32.85 2429 26.79 2018 35.11 19761 58.57 4870 TB-Positive 37 18.14 5148 29.1 2048 22.03 2368 21.22 13496 18.36 19058 TB-Positive 38 54.41 2240 56.99 1503 21.51 2460 38.07 6451 N/A 4257 MTB-Positive 31 21.24 1435 36.22 1456 31.48 2015 24.62 14989 20.32 8328 MTB-Positive 32 20.14 1277 33.79 1296 26.73 2091 24.61 20674 21.21 9507 MTB-Positive 33 22.19 1232 36.95 1289 28.55 2041 22.52 20020 25.86 2935 MTB-Positive 34 21 1317 27.55 4130 27.89 170 22.62 21559 28.2 6440 MTB-Positive 38 20.32 1558 32.39 1324 40.86 1694 22.47 16505 18.3 6935

NTM-Positive 065 N/A 68.7 26.59 4206 N/A 154 32.33 6982 N/A 1167 NTM-Positive 066 N/A 71 30.68 4438 N/A 161 N/A 948 58.09 6773 NTM-Positive 070 N/A 77.6 31.6 2403 26.88 2139 N/A 3257 N/A 1146 NTM-Positive 073 N/A 74.1 26.69 4146 N/A 176 N/A 3345 N/A 4579 NTM-Positive 074 N/A 109 23.3 2809 27.62 1058 N/A 2663 57.59 5043 NTM-Positive 086 N/A 128 19.39 3788 25.23 130 N/A 5753 N/A 347 NTM-Positive 088 N/A 98.2 24.66 4039 30.9 141 N/A 3218 51.74 5601 NTM-Positive 089 N/A 77.7 21.89 2973 27.18 92.1 N/A 5783 N/A -235 NTM-Positive 090 N/A 76.9 20.12 3579 25.87 116 N/A 4401 49.33 8589 NTM-Positive 091 N/A 48.3 54.48 1966 42.07 2744 45.39 12169 N/A 323

TB negative 146 N/A 6.94 N/A 185 28.22 2552 N/A 734 N/A 785 TB negative 147 N/A 5.12 N/A 890 26.96 3397 N/A 796 N/A 853 TB negative 148 N/A 8.28 N/A 203 26.97 2635 N/A 1066 N/A 796 TB negative 149 N/A 6.5 N/A 216 27.55 2948 N/A 943 44.22 20866 TB negative 150 N/A 6.52 N/A 189 26.69 2675 N/A 856 N/A 927 TB negative 105 N/A 1.81 N/A 105 38.61 2049 N/A 5105 41.23 9186 TB negative 106 N/A -0.087 N/A 37.1 30.23 2135 N/A 338 48.37 8730 TB negative 107 N/A 2.25 N/A 60 29.91 2315 N/A 213 N/A 838 TB negative 108 N/A 2.2 N/A 43.2 29.55 2440 N/A 223 N/A 635 TB negative 109 N/A 1.53 N/A 42.7 29.47 2371 N/A 8031 N/A 937

As a result of the test, the primer set of the present invention detected non-specifically for all samples, but the conventional IS6110 primer set showed non-specific results in two non-tuberculous myobacterium samples, and the conventional gyrB primer set detected one tuberculosis sample. It failed and showed 4 non-specific results in non-tuberculous myobacterium specimens and 3 in non-infected specimens.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Primer set for high sensitive multiplex loop-mediated isothermal amplification reaction for detection and identification of Mycobacterium tuberculosis and Nontuberculous mycobacteria <130> DHP20-101 <160> 35 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IS6110_F3 <400> 1 ggtcggaagc tcctatgaca 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IS6110_B3 <400> 2 taggcagcct cgagttcg 18 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> IS6110_FIP <400> 3 agggcttgcc gggtttgatc atgcactagc cgagacga 38 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> IS6110_BIP <400> 4 cggtccatcg aggatgtcga gtcgccgcag tactggtaga 40 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IS6110_FLP <400> 5 ctcggtcttg tataggccgt 20 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> IS6110_BLP1 <400> 6 accgcgcgct gggtcga 17 <210> 7 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> IS6110_BLP1_PROBE2_Cy5 <400> 7 gtcagtgcag gctcccgtgt taggacgagg gtaggaccgc gcgctgggtc ga 52 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> RpoB NTM F3 <400> 8 ccggtcaccg tgctg 15 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RpoB NTM B3 <400> 9 gtarcgcttc tccttgaaga ac 22 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RpoB NTM FIP <400> 10 tgttgtcctt ctccaggtgc tggaccacga gca 33 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> RpoB NTM BIP <400> 11 tggacatcta ccgcaagctg cgttytccar cagggtctgc 40 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> RpoB NTM FLP-2 <400> 12 cggagaaccg aacgctc 17 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RpoB NTM BLP2 <400> 13 ccgccgacca aagagtcagc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RPOB NTM-BLP2_TEX <400> 14 ccgccgacca aagagtcagc 20 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_F3 <400> 15 tccgcacatc ccggtatg 18 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_B3 <400> 16 tctgcagata ggaagggctt 20 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_FIP <400> 17 tggggggcatt gaaggggtga tgagggttct ttgtgctgag 40 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_BIP <400> 18 ctgtggcatc cctgggactg gtgaggaact gggagatcca 40 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_LF <400> 19 atgggccttt ctcccctgc 19 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_LB <400> 20 ggggaaggtt gagcctttac t 21 <210> 21 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_DNA_LAMP_LB_Probe4 HEX <400> 21 cgggcccgta caaagggaac acccacactc cgggggaagg ttgagccttt act 53 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> lamp_PROBE2_QUANCHER <400> 22 cctaccctcg tcctaacacg ggagcctgca ctgac 35 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> lamp_PROBE4_QUANCHER <400> 23 gagtgtgggt gttccctttg tacgggcccg 30 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gyrB_F3 <400> 24 gtgaacaagt acgccaag 18 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gyrB _B3 <400> 25 cacagccttg ttcacaac 18 <210> 26 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> gyrB_FIP <400> 26 tgaccttcac cgagatcaca gctactgaag gacaagga 38 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> gyrB _BIP <400> 27 gccagaccaa gaccaagttg tgttcgttac agaccttct 39 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gyrB _FLP <400> 28 ccttcccgga tattcgtca 18 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gyrB _BLP1 <400> 29 gcaacaccga ggtcaaat 18 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IS6110_F3 <400> 30 ttcgcaattc ggcgttgt 18 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IS6110_B3 <400> 31 atgtcaggtg gttcatcg 18 <210> 32 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> IS6110_FIP <400> 32 tctacttggt gttggctgcg gattcttcgg tcgtggtc 38 <210> 33 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> IS6110_BIP <400> 33 gacctcactg atcgctgccg gatggtcgca gagatcc 37 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IS6110_FLP <400> 34 gagacggtgc gtaagtgg 18 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IS6110_BLP1 <400> 35 actccgaatc gtgctgac 18

Claims (14)

LAMP (Loop-mediated isothermal amplification) primer set for detecting Mycobacterium tuberculosis (MTB), comprising the primers of SEQ ID NOs: 1 to 6.
According to claim 1,
The primer set is a LAMP primer set for detecting Mycobacterium tuberculosis, characterized in that it further comprises a probe consisting of the nucleotide sequence of SEQ ID NO: 7.
3. The method of claim 2,
The probe is 6-carboxyfluorescein (FAM), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) , fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein isothiocyanate), oregon green, Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine) isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes, and any one or more fluorescent substances selected from the group consisting of thiadicarbocyanine LAMP primer set for detecting Mycobacterium tuberculosis, characterized in that it is attached.
LAMP (Loop-mediated isothermal amplification) primer set for detecting non-tuberculosis bacterium (NTM), comprising the primers of SEQ ID NOs: 8 to 13.
5. The method of claim 4,
The primer set is a LAMP primer set for detecting Mycobacterium tuberculosis, characterized in that it further comprises a probe consisting of the nucleotide sequence of SEQ ID NO: 14.
6. The method of claim 5,
The probe is 6-carboxyfluorescein (FAM), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) , fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein isothiocyanate), oregon green, Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine) isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes, and any one or more fluorescent substances selected from the group consisting of thiadicarbocyanine LAMP primer set for non-tuberculosis antibacterial detection, characterized in that it is attached.
A LAMP primer kit for differential detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis, comprising the primer set of claim 1 and the primer set of claim 4.
A primer set for detecting Mycobacterium tuberculosis (MTB) of SEQ ID NOs: 1 to 6, a primer set for detecting Mycobacterium tuberculosis (NTM) of SEQ ID NOs: 8 to 13, an internal control (IC) primer set of SEQ ID NOs: 15 to 20, SEQ ID NO: 7 LAMP kit for tuberculosis differentiation comprising the MTB probe of SEQ ID NO: 14, the NTM probe of SEQ ID NO: 14, and the IC probe of SEQ ID NO: 21.
9. The method of claim 8,
The MTB probe, NTM probe, and IC probe are each independently FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro -6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC ( fluorescein isothiocyanate), oregon green, alexa fluor, JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-XRhodamine) , Tetrachloro-Fluorescein (TET), tertramethylrodamine isothiocyanate (TRITC), 6-carboxytetramethyl-rhodamine (TAMRA), N-(1-Naphthyl) ethylenediamine (NED), cyanine-based dyes and thiadicarbocyanine LAMP kit for tuberculosis diagnosis, characterized in that any one or more fluorescent substances selected from the group consisting of
10. The method of claim 9,
LAMP kit for tuberculosis diagnosis, characterized in that each of the probes is attached with a fluorescent material emitting different wavelengths.
9. The method of claim 8,
LAMP kit for diagnosing tuberculosis, characterized in that the kit further comprises a reagent for performing an amplification reaction
The method of claim 11,
The reagent for performing the amplification reaction is characterized in that it comprises at least one selected from the group consisting of DNA polymerase, dNTPs, and a buffer, LAMP kit for diagnosing tuberculosis.
(1) isolating the genomic DNA (gDNA) of the biological sample;
(2) amplifying the target sequence by using the isolated gDNA as a template and performing an isothermal amplification reaction using the LAMP kit of claim 8; and
(3) detecting the amplified product; information providing method for tuberculosis diagnosis comprising a.
14. The method of claim 13,
The biological sample of step (1) is Mycobacterium tuberculosis and non-tuberculosis, characterized in that at least one selected from the group consisting of sputum, bronchial aspiration and bronchial washing, and body fluid. A method for differential detection of homeobacteria.

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