KR100990756B1 - Probe for Detection of drug resistance of Mycobacterium tuberculosis and the kit - Google Patents

Abstract

The present invention relates to a probe for diagnosing resistance to a drug used as an anti-tuberculosis agent, a kit thereof, and a diagnostic method thereof, and more particularly, a primer capable of amplifying genes related to resistance to rifampin and isoniazid, The present invention relates to a probe capable of detecting resistance related mutations of a gene and a kit for detecting drug resistance of Mycobacterium tuberculosis comprising such primers and probes. It can be used for more effective treatment of tuberculosis patients by diagnosing whether the drug resistance of the drug is faster and more accurate than conventional techniques.

Mycobacterium tuberculosis, Rifampin, isoniazid, drug resistance, mutation, diagnosis

Description

Probe for Detection of drug resistance of Mycobacterium tuberculosis and the kit

The present invention relates to a probe for diagnosing resistance to a drug used as an anti-tuberculosis agent, a kit thereof, and a diagnostic method thereof, and more particularly, a primer capable of amplifying genes related to resistance to rifampin and isoniazid, The present invention relates to a probe capable of detecting a resistance-related mutation of a gene and a drug resistance diagnostic kit for Mycobacterium tuberculosis comprising such primers and probes.

Tuberculosis is a tuberculosis bacterium ( Mycobacterium) that is detected in the droplets of the tuberculosis when a tuberculosis patient coughs. tuberculosis ) , a consumable chronic disease infected by tuberculosis , according to a WHO report, which currently accounts for about 1.9 billion people, or one-third of the world's population, of which 8 million people are infected each year. New tuberculosis (TB) patients are known to develop ( www.who.int/gtb / publications / globrep ).

Tuberculosis is a fundamentally curable disease. The main treatments for tuberculosis include rifampin (RPM), isoniazid (INH), and pyrazinamid (PZA) and etahambutol (EMB). If long-term treatment of the first drug for 6 months and the first drug does not work, chemotherapy using a second drug such as ofloxacin (OFX) or kanamycin (KAN) is used (Tuberculosis). World Health Forum, 14 (4) 438, 1993.In: Ministry of Health and Welfare, Korean Tuberculosis Association, Tuberculosis Control Measures in the 2000s, 1997).

However, the treatment of tuberculosis is caused by multidrug resistant TB (MDR-TB), which is caused by drug-resistant tuberculosis bacteria, particularly rifampin, which has the highest anti-tuberculosis effect, and tuberculosis bacteria that are resistant to children or both drugs. It became very difficult by the advent of it. According to a recent paper, there are now about 50 million MDR-TB patients worldwide, with approximately 275,000 new MDR-TB cases each year, about 3.5% of new TB patients. In this case, the proportion of MDR-TB is estimated to be about 7% (Dye, C., et al., 2002. Worldwide incidence of multidrug-resistant tuberculosis. J. Infect. Dis. 185, 11971202).

Multidrug-resistant tuberculosis does not respond well to treatment, and despite taking medications, it is likely to continue to discharge tuberculosis bacteria and become the source of resistant tuberculosis bacteria, and to spread first-resistant tuberculosis. Therefore, the rapid detection and effective treatment of MDR-TB is essential. According to the World Health Organization (WHO), about 12 new tuberculosis patients develop when one MDR-TB patient is left untreated (Talenti A., Imboden P., Marchesi F., Lowrie D., Cole). S., Colson MJ, Matter L., Schepfer K., Bodmer T. (1993) Detection of rifampin-resistance mutation in Mycobacterium tuberculosis . Lancet 341: 647650).

Rapid and accurate drug sensitivity testing is essential for effective treatment of tuberculosis patients. However, when using the microbiological culture method used in many countries, including Korea, it takes two to three months to isolate the bacteria from the patient and obtain the results of the drug sensitivity test due to the characteristics of the slowly growing tuberculosis bacteria. Because this method is cultured for a long time, it is more likely to be contaminated by other germs, so it is often necessary to retest and requires skilled technicians.

In order to make up for the shortcomings of microbiological methods using solid media, drug sensitivity tests using liquid media have been developed. The BACTEC system and mycobacterium growth indicator tube (MGIT), developed by Becton Dickinson in the United States, inoculate tuberculosis bacteria in liquid media and measure metabolites produced when tuberculosis bacteria begin to grow. However, this method has the disadvantage of limiting the drugs that can be used for the test and nontuberculosis mycobactera (NTM), which can lead to false test results and the need for separate identification of mycobacteria.

Therefore, new methods are needed for rapid and accurate drug sensitivity test. Recently, as the mechanism of drug resistance of Mycobacterium tuberculosis was revealed, researches were actively conducted. As a result, drug resistance of Mycobacterium tuberculosis was found to be caused by an independent mutation in the target of the drug or in a gene region related to the activity of the drug.

For example, the resistance of M. tuberculosis to rifampin has been shown to be encoded by the rpoB gene and associated with amino acid changes in the RNA polymerase ß-subunit, and more than 95% of the rifampin resistant strains contain 81-bp rifampin-resistance determining region ( RRDR) (Mani C, Selvakumar N, et al. J Clin Microbiol. 2001 Aug; 39 (8): 2987-90 .; Telenti A, et al. Lancet. 1993 Mar 13; 341 (8846). ): 647-50.). In addition, mutations associated with ah isoniazid resistance is katG, mabA-inhA, oxyR - ahpC, kasA, ndh (Zhang Y, et al. Infect Immun. 1992 Jun; 60 (6): 2160-5; Banerjee A, et al. Science. 1994 Jan 14; 263 (5144): 227-30.), Generally About 50-95% of strains resistant to isoniazid are katG At the codon 15 site of the gene, 20 -35% of the strains were inhA At the promoter site and 10-15% at the oxyR-ahpC intergenic site (Telenti, A., et al. J. Clin. Microbiol. 1997. 35: 71923; Piatek AS et al. Antimicrob. Agents Chemother. 2000. 44: 10310). Mutations are known to be found (Mokrousov, I., et al. Antimicrob. Agents Chemother. 46: 14171424; Musser, JM, et al. 1996. J. Infect. Dis. 173: 196202; Telenti, A., N. et al. 1997. J. Clin. Microbiol. 35: 719723; Piatek, AS, et al. 2000. Antimicrob.Agents Chemother. 44: 103110) kasA , ndh There is a great deal of difference between regions in terms of isoniazid resistance, and the relationship is still not clear. Based on these findings, the presence of mutations in the gene product targeted by the antituberculosis drug can be used to determine whether the drug is susceptible to Mycobacterium tuberculosis.

Many studies have been conducted on the development of a diagnostic method that can detect the mutation of such drug resistance-related genes, and some research results have been commercialized and used as actual products. Commercially available diagnostic kits include INNO-LiPA Rif., Which detects mutations in the rpoB gene associated with rifampin resistance. TB (Innogenetics, Ghent, Belgium), and more recently katG , inhA , and rpoB The Genotype MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany), which detects the mutations that occur most frequently within, has been commercialized. Genotype MTBDRplus assay and INNO-LiPA Rif. It has the same principle as TB test but has the advantage of being able to diagnose rifampin and isoniazid resistance simultaneously (Johanna Ma¨kinen, et al. J Clin Microbiol., 2006, Feb. 44 (2): 350-352; Doris Hillemann , Et al. J Clin Microbiol., 2005. Aug. 43 (8): 36993703). The basic principle of the two methods is reverse line blot hybridization, which combines wild-type and mutant-type probes bound to nitrocellulose membranes and PCR products of the corresponding genes of Mycobacterium tuberculosis.

However, the prior art uses katG to diagnose isoniazid resistance. Only mutations in the gene and inhA promoter site are detected. As described above, about 50-95% of strains resistant to isoniazid are codon 315, 20 -35% of the strain in the inhA promoter region and 10-15% of the oxyR - ahpC Mutations have been reported in the intergenic region. Thus katG Gene and inhA Diagnosing resistance to isoniazid with only a promoter region mutation may not be able to diagnose about 10-15% resistant strains.

Therefore, the development of rapid diagnosis of anti-tuberculosis drug resistance gene will increase the possibility of appropriate treatment and cure of TB patients, prevent the spread of MDR-TB and reduce the early death.

The present invention solves the above problems and the object of the present invention is to provide a rapid diagnostic method of anti-tuberculosis drug resistance related genes.

The present invention to achieve the above object

Provided are an anti-tuberculosis resistance gene probe having a nucleotide sequence of any one of SEQ ID NOs: 9 to 31.

In the present invention, the anti-tuberculosis agent is preferably rifampin and isoniazid, but is not limited thereto.

In the present invention, the gene probe preferably binds specifically to the katG, inhA, ahpC, and rpoB genes, but is not limited thereto.

In another aspect, the present invention provides an anti-tuberculosis drug resistance diagnostic kit containing a primer capable of amplifying the above-mentioned electron probe and Mycobacterium tuberculosis.

In the present invention, the primer is preferably a primer described in SEQ ID NOs: 1 to 8, but is not limited thereto.

In addition, the kit of the present invention preferably further comprises a label means for detecting a PCR product.

In another aspect, the present invention is to attach the gene probe to the solid support;

b) hybridizing the PCR product of the amplified Mycobacterium tube with the genetic probe; And

c) providing an anti-tuberculosis drug resistance diagnostic method comprising detecting the hybridized product.

In the present invention, the PCR product of Mycobacterium tuberculosis is preferably obtained by the primers described in SEQ ID NOs: 1 to 8, but is not limited thereto.

In the present invention, the detection step is preferably detected using a luminescence or color reaction, but is not limited thereto.

In the present invention, cells were obtained from a sample, nucleic acids were extracted from the cells, and PCR amplification was performed. Methods of extracting nucleic acids from cells and PCR methods were performed through methods well known in the art, such as those described in Molecular cloning (1989) by Sambrook et al.

The nucleic acid amplification method of the present invention used a primer. Generally, the primer is preferably about 10 to 25 bp in length oligonucleotide, but may be longer. The primer is preferably single stranded but may be present in double or single stranded form. It described in the Example.

Primers of the invention were prepared by conventional oligonucleotide synthesis methods, and such synthesis methods are disclosed in US Pat.

Sequences such as primers of the invention can be used to form a double helix optionally with complementary sequences, such as DNA derived from a sample. One skilled in the art can alter the hybridization conditions to change the selectivity of the primer for the target sequence.

In some embodiments, nucleic acids of defined sequences of the invention may be employed in combination with appropriate means such as labels to determine hybridization. Suitable labeling means are various labeling means including fluorescence, radioisotope enzymes or other ligands.

Various template dependent processes are possible to amplify specific gene products present in a given cell sample. One of the most known amplification methods is polymerase chain reaction (PCR), which is described in US Pat. Nos. 4,683,202 and 4,800,159 and the like.

It is generally desirable to separate the amplification product from the template or excess primer or other amplification product in one or another step. For example, the amplification products can be separated using agarose, agarose-acrylamide or PAGE using the standard method described in Molecular cloning (1989) by Sambrook et al.

Chromatography, such as adsorption, partitioning and ion exchange, can also be employed as an effective separation method. These separation methods can be adapted to function in clinical settings to process large volumes of samples, and new methods for separating or detecting thousands of samples at once, FMAT (fluorometric microvolume assay technique), chemiluminescence, and sequence detection systems (Applied) Biosystems) and mass spectroscopy.

Nucleic acids according to the invention will be detected and quantified. In one embodiment the detection can be performed by visual means. Typical visual means method is to dye the gel with ethidium bromide and visualize the bands under UV light. If the amplification product is nucleotides labeled with radiation or fluorescence, the isolated amplification product performs radiosynthesis or fluorescent detection of the inserted radioisotope label.

The present invention includes a kit for carrying out the method. In a non-limiting embodiment the kit of the invention can comprise primers, enzymes for amplification and additional reagents. Thus, the kits will include these one or more reagents in suitable container means. The kits may also include reagents for isolating nucleic acids and purifying amplification products. The components of the kits may be packaged in an aqueous medium or lyophilized form, and suitable container means of the kit include at least one vial, test tube, flask, bottle, etc. into which the components may be placed. If one or more components are present in the kit, they may include other additional containers, such as second, third, etc., where the additional components may be located separately. However, a combination of components may be included in one container.

Hereinafter, the present invention will be described.

The present inventors have attempted to develop a method for quickly and more accurately diagnosing resistance to rifampin and isoniazid.

That is, in order to provide a molecular biological diagnostic method capable of quickly performing the drug sensitivity test of Mycobacterium tuberculosis, which is the main object of the present invention, genes that are targets of drugs of Mycobacterium tuberculosis bacterium showing resistance and sensitivity to each drug, rpoB, katG and mabA -inhA, oxyR- ahpC Based on the results of sequencing of the site, wild-type and mutant probes were prepared to distinguish drug-resistant and sensitive bacteria. Therefore, it was confirmed that the use of such a probe can quickly diagnose resistance to rifampin and isoniazid, thereby completing the present invention.

Therefore, using the kit of the present invention, the susceptibility test for the tuberculosis rifampin and isoniazid can be carried out quickly and accurately, thus contributing to the effective treatment of tuberculosis patients, thereby helping to effectively block the spread of multidrug-resistant tuberculosis bacteria. Will be.

The present invention provides a drug resistance diagnostic kit for Mycobacterium tuberculosis comprising a primer capable of amplifying resistance-related genes for rifampin and isoniazid, a major anti-tuberculosis agent, and a probe for detecting a resistance-related mutation of the gene. In this case, it is possible to detect whether each gene is mutated and through this, it is possible to quickly and accurately diagnose the resistance to rifampin and einsoniazid. Therefore, the present invention will contribute to the effective treatment of tuberculosis patients.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. will be.

1 is a flowchart schematically illustrating a process of carrying out the present invention. The present invention amplifies four sites known to be related to rifampin and isoniazid resistance as shown in the flowchart through multiplex PCR, and amplifies the wild type and mutants that can determine the rifampin and isoniazid resistance bound to the membrane. React with the type probe and wash to remove nonspecific binding PCR products. In addition, it processes enzymes that can show the binding of probes and PCR products as visible signals, and finally transfers these signals to hypersensitive films or image files for analysis.

Example 1 : Obtain DNA of Mycobacterium tuberculosis

Strains resistant to rifampin and isoniazid and sensitive strains were cultured in aerobic state for 3 weeks in Ogawa medium, and then the colonies of tuberculosis formed were collected in a loop and 400 μl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Loosen well. And it is reacted for one hour at 80 ℃ to kill the tuberculosis bacteria. 50 μl of lysozyme (10mg / ml) was added to the well, followed by reaction overnight at 37 ° C. Add 5 μl of 10 mg / ml proteinase K (Sigma Chem. Co., USA) and 70 μl of 10% (w / v) SDS (Sodium dodexyl sulfate, Sigma Chem. Co., USA) and mix well. The reaction was carried out at 65 ° C. for 10 minutes. Add 100 μl of 5M NaCl (Sodium chloride, Ducksan, Korea), mix, add 100 μl of CTAB (Cetyl Trimethyl Ammonium Bromide, Sigma Chem. Co., USA) preheated at 65 ° C, mix well, and mix for 10 minutes at 65 ° C. Reacted. 700 μl of Chloroform: isoamylalcohol (= 24: 1 (v / v)) was added well, followed by centrifugation. Only the supernatant containing DNA was transferred to a new tube, 450 μl of isopropanol (Merck) was added and mixed, and the DNA was precipitated at −20 ° C. for 30 minutes. After centrifugation at 4 ° C and 12,000 rpm for 30 minutes, the supernatant was decanted and 1 ml of 70% ethanol was added and the tube was inverted about 5 times. After centrifugation again at 4 ° C. for 10 minutes, the supernatant was discarded and dried for 30 minutes, and DNA was dissolved in 50 μl of TE. After confirming the concentration, the dissolved DNA was diluted to 1ng / μl and used for PCR.

Example 2: Preparation of the M. tuberculosis katG, inhA, ahpC, rpoB Gene amplification

From genomic DNA of M. tuberculosis showing resistance and sensitivity to rifampin was separated in Example 1 and sub isoniazid katG, inhA, ahpC, rpoB In order to simultaneously amplify the gene region, primers capable of amplifying each gene were designed and biotin was labeled at the 5 'end of the reverse primer to visualize the signal later (see Table 1).

gene primer primer  Sequence Tm GC (%) Length rpoB
 470-F-F  GCG TCG GTC GCT ATA AGG T 55 58 19 SEQ ID NO: 1  614- FR  * ACG TCG CGG ACC TCC A 65 71 19 SEQ ID NO: 2 katG
katP1 -One CAC ACT TTC GGT AAG ACC CA 52 60 20 SEQ ID NO: 3 katP2 -2 * GAA ACT GTT GTC CCA TTT CG 52 65 20 SEQ ID NO: 4 inhA
TB92 -One CGT AGG GCG TCA ATA CA 53 45 20 SEQ ID NO: 5 TB93 -One * CCT GGT GCT CTT CTA CC 59 60 20 SEQ ID NO: 6 ahpC
TB91 -One GTC GAC TGG CTC ATA TC 55 50 20 SEQ ID NO: 7 TB90 -One * TGA TCG CCA ATG GTT AG 51 40 20 SEQ ID NO: 8

The table katG, inhA, ahpC, rpoB A primer for amplifying the gene region is shown, and '*' in the table represents a primer labeled with Biontin.

5 μl of each DNA obtained above was used as a template, and 4 pairs of primers of SEQ ID NOS: 1-8, dNTP, and Taq DNA polymerase were used as PCR equipment (Thermal cycler, GeneAmp PCR).  PCR was performed on System 2700 (95 ° C 5 minutes, 95 ° C 30 seconds, 54 ° C 1 minute 30 seconds, 72 ° C 1 minute, 35cycles, 72 ° C 10 minutes), katG (189bp), inhA ( 442 bp), ahpC (250 bp) and rpoB (470 bp) gene sites were amplified.

Example 3 : Diagnosis of drug resistance of Mycobacterium tuberculosis using a probe capable of diagnosing rifampin and isoniazid resistance (luminescence)

Of M. tuberculosis showing resistance and sensitivity to rifampin and isoniazid O obtained in Example 1 katG, inhA, ahpC, rpoB Based on the nucleotide sequence of the gene region, a probe that can specifically bind to each wild type and mutant sequence was designed and manufactured by binding an amine group to the 5 'end of each probe to bind the probe to the membrane (see Table 2)

Probe Probe sequence  Myc  GACGTCGTCGCCACCATCGA  Mycobacteria SEQ ID NO: 9  TB  GCTGCATGTCGGCGAGC M. tuberculosis SEQ ID NO: 10 rpoB WT1  AGCCAGCTGAGCCAATTC  509-514 SEQ ID NO: 11 rpoB WT2  ATGGACCAGAACAACCCG  515-520 SEQ ID NO: 12 rpoB WT3  CGGCTGTCGGGGTTGACC  521-525 SEQ ID NO: 13 rpoB WT4  TTGACCCACAAGCGCCGA  524-529 SEQ ID NO: 14 rpoB WT5  CTGTCGGCGCTGGGGC  530-534 SEQ ID NO: 15 rpoB MT1-1  CTGTTGGCGCTGGGGC  531TTG SEQ ID NO: 16 rpoB MT1-2  AAATGTCGGCGCCGGGGCC  533CCG SEQ ID NO: 17 rpoB MT2-1  AATGGTCCAGAACAACCCG  516GTC SEQ ID NO: 18 rpoB MT2-2  TTCATGTACCAGAACAACCCG  516 TAC SEQ ID NO: 19 rpoB MT3  GCTGAGCCCAGGCATGGAC  513CCA SEQ ID NO: 20 katG 315 WT  CACCAGCGGCATCGAG  315AGC SEQ ID NO: 21 katG 315 MT  ATCACCACCGGCATCGAG  315AAC SEQ ID NO: 22 inhA 15UPS WT  CGCGGCGAGACGATAGG SEQ ID NO: 23 inhA 15UPS MT  CGCGGCGAGATGATAGG  15C → T SEQ ID NO: 24 inhA 8UPS WT  AAAGATAGGTTGTCGGGGTGACT SEQ ID NO: 25 inhA 8UPS MT  GACGATAGGCTGTCGGGG  8T → C SEQ ID NO: 26 ahpC WT1  GCCGATAAATATGGTGTGATATATCA SEQ ID NO: 27 ahpC WT2  CCTTTGCCTGACAGCGACTT SEQ ID NO: 28 ahpC WT3  CACGGCACGATGGAATGTC SEQ ID NO: 29 ahpC WT4  GCAACCAAATGCATTGTCCGC SEQ ID NO: 30 ahpC WT5  TTTGATGATGAGGAGAGTCATGC SEQ ID NO: 31

The table shows a probe capable of diagnosing the resistance of rifampin and isoniazid to Mycobacterium tuberculosis.

Subsequently, a reverse blot hybridization method was performed using each of the electroproduced probes. First, a 16% biodegradation membrane (Biodyne C binding membrane, Pall Biosupport, MI, USA) was used. (w / v) carboxyl group on the membrane surface by immersion in 10 ml of EDAC (1-ethyl-3-dimethyl-aminopropyl- cardiimide, Sigma Chem., Co., USA) solution for 15 minutes at room temperature ) Was activated, washed with distilled water and mounted in a blot (MN45 minblotter, Immunetics, USA).

Each probe prepared to include an amine group (amino group) at the 5 'end was diluted in 500 mM NaHCO ₃ (pH 8.4) so that the final volume was 150 µl, filled in an electric blot, and then 70 minutes at room temperature. In reaction, the electrical probe was coupled to the electrical membrane. After the reaction was completed, the solution containing the probe was removed, the membrane was removed from the electric blot, and then immersed in 100 ml of 0.4N NaOH solution at room temperature for 9 minutes to prevent the probe from further binding. The carboxyl group on the membrane surface was inactivated. It was washed for 5 minutes at 60 ° C. with 2 × SSPE containing 0.1% (w / v) SDS. The electrowashed membrane was remounted in the blot to be perpendicular to the direction in which the probe was coupled.

Meanwhile, 45 μl of the PCR product of Mycobacterium tuberculosis amplified in Example 2 was diluted with 2 × SPPE / 0.1% (w / v) SDS solution. And then added to an electric blot. Then, hybridization reaction was performed at 50 ° C. for 60 minutes. After the electrical dilution was removed, the membrane was separated and washed twice at 65 ° C. with 2 × SPPE / 0.5% (w / v) SDS solution for 15 minutes. Alkaline phosphatase-labeled streptavidin, Boehringer Mannheim, diluted 1: 2000 (v / v) in 2 × SPPE / 0.5% (w / v) SDS solution on electro washed membranes Germany) and reacted for 40 minutes at 42 ° C., which was washed twice with 2 × SPPE / 0.5% (w / v) SDS solution at 42 ° C. for 10 minutes and twice with 2 × SPPE solution at room temperature for 5 minutes. . Labeled biotin was detected by application to a substrate solution (Gene Images CDP-star ™ detection kit, Amersham Bioscience, UK) in response to alkaline phosphatase (see FIG. 1). 1 is a photograph showing the results of the diagnosis of rifampin and isoniazid resistance of Mycobacterium tuberculosis using a reverse blot hybridization method.

Example 4 : Diagnosis of drug resistance of Mycobacterium tuberculosis using a probe capable of diagnosing rifampin and isoniazid resistance (coloring method)

Specific binding to wild type and mutant sequences based on the nucleotide sequences of katG, inhA, ahpC, and rpoB gene regions of Mycobacterium tuberculosis bacillus showing resistance and sensitivity to rifampin and isoniazid in the same manner as in Example 3. A reverse blot hybridization method using a probe designed to be performed was performed. First, a reverse blot hybridization membrane (Biodyne C binding membrane, Pall Biosupport) was performed in the same manner as in Example 3. , MI, USA). The PCR product of Example 2 was diluted with 2 × SPPE / 0.1% (w / v) SDS solution on the prepared membrane, heated at 100 ° C. for 10 minutes, and rapidly cooled to denature the PCR product at 50 ° C. for 60 minutes. Hybridization reactions were performed. The dilution was removed and then washed twice with 2 × SPPE / 0.5% (w / v) SDS solution at 65 ° C. for 15 minutes. Alkaline phosphatase-labeled streptavidin, Roche Applied Science diluted 1: 2000 (v / v) in 2 × SPPE / 0.5% (w / v) SDS solution on electro washed membranes , Germany) for 40 minutes at 42 ℃. The reaction membrane was washed twice with TBS solution (pH7.5) at room temperature for 10 minutes. NBT / BCIP, Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt in 67% DMSO (v / v), Roche Applied Science, Germany] was diluted 1:50 with TBS solution of pH 9.5 and reacted to the membrane at room temperature for 10-30 minutes to detect labeled biotin on the PCR product bound to the probe (see FIG. 1). After the color reaction was completed, the color reaction was removed and tertiary distilled water was added to stop the color reaction. 4 is a photograph showing the results of diagnosing rifampin and isoniazid resistance of Mycobacterium tuberculosis using a reverse blot hybridization method (coloring method).

1 is a schematic flowchart of the present invention. That is, ① the amine-labeled probe is bound to the membrane. ② the target gene is amplified with a biotin-labeled primer and then reacted with the probe bound to the membrane. ③ Reacts with Streptavidin-conjugated alkaline phosphatase. ④ Detects defects of probe and amplified target gene by treating substrate of Alkaline phosphatase.

Figure 2 is a picture of the electrophoresis of the PCR product for diagnosing the resistance to rifampin and isoniazid. In the photograph, M: 100bp DNA Ladder (Bionia, Daejeon), Lane1-30: PCR amplification product of clinically isolated tuberculosis strain, P: PCR product of standard tuberculosis strain, N: PCR negative control.

Figure 3 is a photograph showing the results of diagnostic diagnosis of Mycobacterium tuberculosis resistance to rifampin and isoniazid using a reverse blot hybridization method. In the results table below, R means drug resistance and S means drug sensitivity.

Figure 4 is a photograph showing the results of the diagnostic diagnosis of Mycobacterium tuberculosis resistance to rifampin and isoniazid using a reverse blot hybridization method. In the results table below, R means drug resistance and S means drug sensitivity.

<110> Molecules and Diagnostics Inc. <120> Probe for Detection of drug resistance of Mycobacterium          tuberculosis and the kit <160> 31 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 470-F-F <400> 1 gcgtcggtcg ctataaggt 19 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 614-FR <400> 2 acgtcgcgga cctcca 16 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> katP1-1 <400> 3 cacactttcg gtaagaccca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> katP2-2 <400> 4 gaaactgttg tcccatttcg 20 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TB92-1 <400> 5 cgtagggcgt caataca 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TB93-1 <400> 6 cctggtgctc ttctacc 17 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TB91-1 <400> 7 gtcgactggc tcatatc 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TB90-1 <400> 8 tgatcgccaa tggttag 17 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myc <400> 9 gacgtcgtcg ccaccatcga 20 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TB <400> 10 gctgcatgtc ggcgagc 17 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rpoB WT1 <400> 11 agccagctga gccaattc 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rpoB WT2 <400> 12 atggaccaga acaacccg 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rpoB WT3 <400> 13 cggctgtcgg ggttgacc 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rpoB WT4 <400> 14 ttgacccaca agcgccga 18 <210> 15 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> rpoB WT5 <400> 15 ctgtcggcgc tggggc 16 <210> 16 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> rpoB MT1-1 <400> 16 ctgttggcgc tggggc 16 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rpoB MT1-2 <400> 17 aaatgtcggc gccggggcc 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rpoB MT2-1 <400> 18 aatggtccag aacaacccg 19 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rpoB MT2-2 <400> 19 ttcatgtacc agaacaaccc g 21 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rpoB MT3 <400> 20 gctgagccca ggcatggac 19 <210> 21 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> katG 315 WT <400> 21 caccagcggc atcgag 16 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> katG 315 MT <400> 22 atcaccaccg gcatcgag 18 <210> 23 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> inhA 15UPS WT <400> 23 cgcggcgaga cgatagg 17 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> inhA 15UPS MT <400> 24 cgcggcgaga tgatagg 17 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> inhA 8UPS WT <400> 25 aaagataggt tgtcggggtg act 23 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> inhA 8UPS MT <400> 26 gacgataggc tgtcgggg 18 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ahpC WT1 <400> 27 gccgataaat atggtgtgat atatca 26 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ahpC WT2 <400> 28 cctttgcctg acagcgactt 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ahpC WT3 <400> 29 cacggcacga tggaatgtc 19 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ahpC WT4 <400> 30 gcaaccaaat gcattgtccg c 21 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ahpC WT5 <400> 31 tttgatgatg aggagagtca tgc 23  

Claims (10)

An anti-tuberculosis resistance gene probe composition consisting of a probe having a nucleotide sequence of SEQ ID NOs: 9 to 31. According to claim 1, wherein the anti-tuberculosis agent is rifampin and isoniazid anti-tuberculosis resistance diagnostic gene probe composition, characterized in that. The gene probe composition of claim 1, wherein the gene probe specifically binds to katG, inhA, ahpC, and rpoB genes. An anti-tuberculosis drug resistance diagnostic kit comprising the gene probe composition of any one of claims 1 to 3 and a primer composition capable of amplifying Mycobacterium tuberculosis. The anti-tuberculosis drug resistance diagnostic kit according to claim 4, wherein the primer composition is composed of the primers set forth in SEQ ID NOs: 1-8. According to claim 4, The kit is anti-tuberculosis drug resistance diagnostic kit, characterized in that it further comprises a label means for detecting a PCR product. delete delete delete delete

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