KR102274809B1 - Medium composition for detecting drug resistant mycobacterium tuberculosis and kit for detecting drug resistant mycobacterium using it - Google Patents

Abstract

Disclosed in the present invention are a medium composition for detecting drug resistant Mycobacterium tuberculosis, and a kit for detecting drug resistant Mycobacterium tuberculosis by using same. The medium composition and the kit according to the present invention do not inhibit the proliferation (growth) of Mycobacterium tuberculosis, allow the boundaries of colonies of the proliferated drug resistant Mycobacterium tuberculosis to be clear, and allow the color of colonies of Mycobacterium tuberculosis to be in complementary contrast with that of the medium composition, thereby enabling the colonies of Mycobacterium tuberculosis to be easily, conveniently and accurately counted and detected with the naked eye.

Description

A medium composition for detecting drug-resistant Mycobacterium tuberculosis and a kit for detecting drug-resistant Mycobacterium tuberculosis using the same {Medium composition for detecting drug resistant mycobacterium tuberculosis and kit for detecting drug resistant mycobacterium using it}

The present invention relates to a medium composition for detecting drug-resistant Mycobacterium tuberculosis and a kit for detecting drug-resistant Mycobacterium tuberculosis using the same, and more particularly, to include a specific pigment at a specific concentration to easily and accurately detect drug-resistant Mycobacterium tuberculosis without inhibiting the proliferation of Mycobacterium tuberculosis. It relates to a medium composition and a kit for detecting drug-resistant Mycobacterium tuberculosis comprising the medium composition.

Tuberculosis is a class 2 statutory infectious disease that needs to be reported and isolated within 24 hours when it occurs or spreads in consideration of the possibility of transmission. According to the WHO’s Global Tuberculosis Report 2018, as of 2017, Korea had a tuberculosis incidence rate of 70 per 100,000 people and a mortality rate of 70 people per 100,000 people. The number is 5, the highest among 35 OECD member countries, and is significantly higher than the OECD average of 11.1 cases of tuberculosis per 100,000 and the mortality rate of 0.9 cases per 100,000 people. Tuberculosis is initially checked for infection through chest X-ray and sputum test, and if infected, antibiotics are prescribed for treatment. After that, depending on the progress, the first, second, or third stage antibiotics are taken. In order to take antibiotics for each stage, it is necessary to determine whether there is resistance to each antibiotic.

The treatment of tuberculosis is the treatment of drug-resistant Mycobacterium tuberculosis, in particular, multidrug resistant tuberculosis (MDR-TB) caused by rifampin or isoniazid, which has the highest antituberculosis effect, or Mycobacterium tuberculosis resistant to both drugs. It became very difficult by the appearance. It is known that there are 50 million MDR-TB patients worldwide, and about 275,000 new MDR-TB patients occur every year, which is about 3.5% of new tuberculosis patients. The rate is estimated to be about 7%, and extensively drug-resistant tuberculosis (XDR-TB), the most resistant among multidrug-resistant tuberculosis, has been reported in academia as less than 50% of the treatment results ( Dye, C., 2002. Worldwide incidence of multidrug-resistant tuberculosis. J. Infect. Dis. 185,1197-1202).

Since treatment delay caused by drug-resistant Mycobacterium tuberculosis leads to regional spread of drug-resistant Mycobacterium tuberculosis, it is very important to determine whether Mycobacterium tuberculosis is drug-resistant during the initial diagnosis of tuberculosis.

Recently, drug susceptibility tests based on molecular biological testing methods have been developed and their use is increasing, and the WHO recommends rapid testing using molecular biological-based rapid resistance diagnostics and initiation of treatment accordingly. However, since the types and number of mutations of resistance-inducing genes for each currently known antituberculosis drug are diverse, it is not easy to conduct the test at once by conventional methods such as PCR, line probe assay, and real-time PCR. Therefore, the Mycobacterium tuberculosis drug resistance test used as a standard method so far is a culture-based method that judges whether or not the Mycobacterium tuberculosis colony grows using a medium containing antibiotics. However, the color of the colony of Mycobacterium tuberculosis and the medium was off-white, so it was not easy to identify with the naked eye, so it was not easy to determine whether or not to multiply, and the accuracy of the judgment results tended to be low.

Therefore, it has been desired to develop a medium composition that can easily and accurately detect drug-resistant Mycobacterium tuberculosis because the growth of Mycobacterium tuberculosis is made well and it is easy to identify with the naked eye.

Accordingly, the present inventors have continued research to meet the needs of the prior art, and as a result of using a medium composition containing a specific pigment at a specific concentration, a colony of drug-resistant Mycobacterium tuberculosis can be easily, conveniently and accurately detected with the naked eye. It was confirmed that it is possible to complete the present invention.

Accordingly, it is an object of the present invention to provide a medium composition for detecting drug-resistant Mycobacterium tuberculosis, which can be easily, conveniently and accurately detected with the naked eye.

Another object of the present invention is to provide a kit for detecting drug-resistant Mycobacterium tuberculosis comprising the medium composition.

Another object of the present invention is to provide a method for detecting drug-resistant Mycobacterium tuberculosis using the medium composition or the kit.

In order to achieve the above object, the present invention provides a medium composition for detecting drug-resistant Mycobacterium tuberculosis comprising food color blue No. 1 as an active ingredient.

In the present invention, food color blue No. 1 is 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3-sulfonatebenzyl)amino]phenyl](2-sulfonatephenyl) methylene]-2,5-cyclohexadienylidene]ammoniomethyl]benzenesulfonate disodium (C37H31O9N2S3Na2) 85.0% (w/w) or more.

In the medium composition of the present invention, food color blue No. 1 may be included in an amount of 0.01 to 0.03 g/L, preferably 0.0125 to 0.025 g/L, and most preferably 0.0125 g/L. When the food color blue No. 1 is contained at less than 0.01 g/L, the color of the medium composition is light and it is not easy to detect the proliferated Mycobacterium tuberculosis colony. If it contains more than 0.03 g/L, the color of the medium composition is too dark. Visual detection of colonies is not easy, and the growth (growth) of Mycobacterium tuberculosis may be inhibited.

As the medium in the present invention, a solid medium for culturing Mycobacteria may be used, preferably Middlebrook 7H10 agar medium, LJ medium, or the like.

The medium composition of the present invention may further include a growth supplement (enrichment) if necessary, and preferably, a Middlebrook OADC (oleic albumin dextrose catalase) growth supplement may be further included.

The medium composition of the present invention may contain an antibiotic (anti-tuberculosis agent) having anti-tuberculosis activity. The antibiotic is ethambutol (Ethambutol), isoniazid (Isoniazid), rifampicin (Rifampicin), rifabutin (Rifabutin), amikacin (Amikacin), kanamycin (Kanamycin), capreomycin (Capreomycin), streptomycin (Streptomycin), (Levofloxacin), moxifloxacin, prothionamide, cycloserine (Cycloserine), pars (P-aminosalicylic acid; P-aminosalicylicacid), bedaquiline (Bedaquiline), linezolid (Linezolid) ), and may be one selected from the group consisting of delamanid.

In the medium composition of the present invention, the antibiotic may be added at a concentration of 0.2 to 30 ug/ml depending on the type.

When the medium composition of the present invention is used, the boundaries of the colonies (colonies) of the drug-resistant Mycobacterium tuberculosis are clear and the colony color of Mycobacterium tuberculosis is contrasted with the medium composition, so that the colonies of Mycobacterium tuberculosis proliferated easily, conveniently and accurately with the naked eye are counted and detected. can (FIGS. 1A and 1B). In the case of a conventional medium composition, it was difficult to count Mycobacterium tuberculosis colonies with the naked eye because the boundaries of the colonies of Mycobacterium tuberculosis proliferated were unclear (typically 20-30%) because the color was similar to that of the Mycobacterium tuberculosis colony (FIG. 4). In contrast, the medium of the present invention In the case of the composition, there were few cases in which the boundaries of the Mycobacterium tuberculosis colony were unclear (less than about 5%) ( FIGS. 1A , 5A to 6E ).

According to another object of the present invention, the present invention provides a kit for detecting drug-resistant Mycobacterium tuberculosis comprising the medium composition.

The case of the kit of the present invention preferably has a structure in which 6-wells having a diameter of 35 mm are mounted on a fixed plate (6-well plate) ( FIGS. 2A and 2B ). As shown in FIG. 2B , a 6-well plate can be tested for resistance to four antibiotics at the same time, including a 6-well.

In addition, according to the use of the medium composition of the present invention, the diameter of the well of the 6-well plate can easily detect the colonies of drug-resistant Mycobacterium tuberculosis even at 1/6 the size of a conventional well (10 pie) (FIG. 3).

In the kit of the present invention, 6-well may consist of two control groups (antibiotic-free medium composition) and up to 4 test groups (antibiotic-added medium composition). Preferably, the test group may be a combination of a medium composition to which the following antibiotics are added ( FIGS. 5A to 6E ):

1) Oral first-line antituberculosis: ethambutol (5.0 ug/ml), isoniazid (0.2 ug/ml), rifampicin (1.0 ug/ml) and rifabutin (0.5 ug/ml)

2) Secondary anti-tuberculosis agents for injection: amikacin (4.0 ug/ml), kanamycin (5.0 ug/ml), capreomycin (10.0 ug/ml), streptomycin (2.0 ug/ml)

3) Quinoline and primary and secondary high-dose anti-tuberculosis agents: levofloxacin (1.0 ug/ml), moxifloxacin (0.5 ug/ml), isoniazid (1.0 ug/ml), streptomycin (10.0 ug/ml)

4) Secondary oral anti-tuberculosis drugs: Prothionamide (5.0 ug/ml), Cycloserine (30.0 ug/ml), PAS (P-aminosalicylic acid; 2.0 ug/ml)

5) New drug antibiotics: vedaquiylin (0.25 ug/ml), linezolid (1.0 ug/ml), and delamanid (0.016 ug/ml).

When the kit of the present invention is used, the colonies of the proliferated drug-resistant Mycobacterium tuberculosis can be easily, conveniently, and accurately counted and detected with the naked eye.

According to another object of the present invention, there is provided a method for detecting drug-resistant Mycobacterium tuberculosis using the medium composition or the kit. the method

i) collecting a sample of sputum from an individual and culturing enrichment; and

ii) inoculating and culturing the enriched culture medium in the kit containing the medium composition.

In the present invention, "individual" means a tuberculosis patient or a person suspected of tuberculosis.

In the above method, 'medium composition' and 'kit' are as defined above.

In the present invention, 'enrichment culture' refers to culturing a sputum sample in Ogawa tetragonal solid medium for 3 to 4 weeks and then using a vortex stirrer or automatic sterilizer to prepare a bacterial solution from the proliferated Mycobacterium tuberculosis colony. Not limited.

In the present invention, 'enrichment culture solution' refers to the bacterial solution produced through the enrichment culture.

In step ii), inoculation is carried out by diluting the enriched culture medium according to the concentration of the standard Mycobacterium tuberculosis (3.0x10 ⁸ ㎖/Approximate Bacterial Suspension) solution, and adding 20-30 μl to the medium surface at room temperature so that the bacterial solution is absorbed into the medium.

Culturing in step ii) may be performed by culturing for 2 to 3 weeks at a temperature of 35 to 37° C. and 5 to 10% CO _{2 atmospheric conditions.}

In the method of the present invention, when the number of colonies of Mycobacterium tuberculosis in the test group is 20 or more, or when the number of colonies of Mycobacterium tuberculosis increases by 1% or more compared to the positive control group, drug resistance is determined.

According to the method of the present invention, the colonies of the proliferated drug-resistant Mycobacterium tuberculosis can be easily, conveniently, and accurately counted and detected with the naked eye.

The medium composition according to the present invention does not inhibit the growth (growth) of Mycobacterium tuberculosis, the boundaries of the colonies of the proliferated drug-resistant Mycobacterium tuberculosis are clear, and the color of the colony of Mycobacterium tuberculosis is in contrast to the complementary color of the medium composition, so that it is easily, conveniently and accurately with the naked eye. It can be detected by counting colonies.

In addition, the well diameter used in the medium composition, kit, and method using the same of the present invention can easily detect a colony of drug-resistant Mycobacterium tuberculosis even with 1/6 the size of a conventional well. In addition, the kit according to the present invention is composed of a 6-well plate and can be tested by combining up to four drugs in each antituberculous drug prescription step, so that the test period and cost can be significantly reduced.

Therefore, according to the medium composition, the kit, and the method using the same according to the present invention, it is possible to easily, simply and accurately diagnose whether an individual has drug-resistant tuberculosis.

1A and 1B are photographs showing the Mycobacterium tuberculosis colonies proliferated in the medium composition according to the present invention and their counting. Arrows are indicated to count Mycobacterium tuberculosis colonies, and a total of 50 colonies were counted with 10 yellow, 10 green, 10 light purple, 10 deep yellow, 7 purple, and 3 red.
2A is a perspective view showing an example of a case (6-well plate) of the kit of the present invention.
Figure 2b is a photograph showing an example of the kit (6-well plate) of the present invention containing the medium composition of the present invention.
3 is a photograph comparing the well size (blue circle) on the kit case of the present invention in a conventional well plate for testing the drug resistance of Mycobacterium tuberculosis.
4 is a photograph of the culture test result of the comparison kit.
5A to 5E are photographs of the results of a drug resistance culture test of Mycobacterium tuberculosis using the kit of the present invention (in the absence of drug resistance).
6A to 6E are photographs of the results of a culture test for Mycobacterium tuberculosis using the kit of the present invention (when drug resistance is detected).

The present invention is further illustrated by the following examples. These examples are for the purpose of illustrating the present invention, and the scope of the present invention should not be limited thereto.

Example 1. Preparation of medium composition and kit

900 ml of distilled water and 5 ml of glycerol were added to 19 g of Middlebrook 7H10 agar medium powder having the composition shown in Table 1 below, mixed well at 100 rpm, sterilized at 121°C for 15 minutes, cooled to 50-55°C, and sterilized As a result, a medium was prepared by mixing 100 ml of Middlebrook OADC growth supplement (Enrichment) as shown in Table 1 below. Then, 0.5 g of Food Color Blue No. 1 (ES Food Co., Ltd.) was dissolved in 40 ml of distilled water, and 1 ml was added to 1 L of the medium (concentration of Food Color Blue No. 1 0.0125 g/L) to prepare a medium composition. prepared.

Middlebrook 7H10 agar medium 0.5 g Ammonium Sulfate
1.5 g Monopotassium Phosphate
Disodium Phosphate 1.5 g
Sodium Citrate 0.4 g
Magnesium Sulfate 25.0 mg
Calcium Chloride 0.5 mg
Zinc Sulfate 1.0 mg
Copper Sulfate 1.0 mg
L-Glutamic Acid (sodium salt) 0.5 g
Ferric Ammonium Citrate 0.04 g
Pyridoxine Hydrochloride 1.0 mg
Biotin 0.5 mg
Malachite Green 250.0 μg
Agar 15.0g Middlebrook OADC Enrichment Sodium Chloride 8.5 g
Dextrose 20.0 g
Bovine Albumin (Fraction V) 50.0 g
Catalase 0.03 g
Oleic Acid 0.6 mL

After each addition of the antibiotics and concentrations shown in Table 2 to the test group medium composition, 4 ml of each medium composition was dispensed into each well of the 6-well flight kit case of the present invention (Fig. 2a), and the control wells Each of the medium composition was dispensed by 4ml, to prepare 5 kits according to the present invention as shown in Table 2 below (Fig. 2b).

control test group test group I positive control 5.0 μg/ml
Ethambutol 0.2 μg/ml
Isoniazid negative control 1.0 μg/ml
Rifampicin 0.5 μg/ml
Rifabutin II positive control 4.0 μg/ml
Amikacin 5.0 μg/ml
Kanamycin negative control 10.0 μg/ml
Capreomycin 2.0 μg/ml
Streptomycin Ⅲ positive control 1.0 μg/ml
Levofloxacin 0.5 μg/ml
Moxifloxacin negative control 1.0 μg/ml
Isoniazid 10.0 μg/ml
Streptomycin IV positive control 5.0 μg/ml
Prothionamide 30.0 μg/ml
Cycloserine negative control 2.0 μg/ml
P-aminosalicylicacid - Ⅴ positive control 0.25 μg/ml
Bedaquiline 1.0 μg/ml
linezolid negative control 0.016 μg/ml
Delamanid -

Antibiotics were not added to the positive control group and the negative control group, and only the medium composition according to the present invention was added. Upon subsequent examination, the positive control group is a group inoculated with Mycobacterium tuberculosis solution, and the negative control group is a group that is not inoculated with Mycobacterium tuberculosis solution. The test group is a group to which antibiotics have been added, and - indicates that the wells of the kit are empty.

For comparison, the conventional medium composition prepared as above was prepared with Middlebrook 7H10 agar medium and Middlebrook OADC growth supplement (Enrichment) except for food coloring, and 4ml in each well of the 6-well flight kit case of the present invention A comparison kit was prepared by dispensing each.

Example 2: Drug resistance detection test

The Mycobacterium tuberculosis drug resistance detection test was performed for the 5 kits (I to V) prepared in Example 1.

Specifically, sputum from tuberculosis patients (while hospitalized at Masan Hospital) was collected and cultured in Ogawa slope solid medium (Bandio Co., Ltd.) for 3 weeks at 37°C. Then, the cultured Mycobacterium tuberculosis colonies were collected in a loop and treated with McCartney's disease. After carefully dispersing and transferring over the glass beads with 50 μl of 10% Tween 80, close the bottle cap and shake for 20 seconds with a vortex stirrer to crush the fungal mass, and mix by rotating the bottle containing the glass beads left and right. If the fungal mass was attached to the bottle neck, the beaded bottle was further laid down to mix, and after shaking with a vortex stirrer, it was left at room temperature for 10 minutes to prepare a bacterial solution. The Mycobacterium tuberculosis solution for inoculation was prepared by diluting the bacterial solution according to the concentration of McFarland 1.0 standard bacterial solution (3.0x10 ^{8 ㎖/Approximate Bacterial Suspension).}

25 μl of the Mycobacterium tuberculosis solution was inoculated on the medium side of the positive control group and test group of the kit using a micropipette, respectively, so that the bacterial solution was absorbed into the medium, and then the kit cover was covered and put into a _{CO 2 permeable polyethylene bag.} 37° C., 5-10% CO ₂ Atmospheric conditions, with the medium side facing up, were cultured for 3 weeks. If sufficient growth was observed in the test medium compared to the positive control group, it was judged as resistant (in case of 20 or more colonies or 1% or more growth compared to the positive control medium).

For comparison, the Mycobacterium tuberculosis solution was inoculated and cultured in the same manner as above in the comparison kit prepared in Example 1.

The culture results are shown in Fig. 4 (comparative kit), Figs. 5a to 5e (test kit - when drug resistance is not confirmed) and Figs. 1a, 1b, 6a to 6e (test kit - when drug resistance is confirmed) shown in

4 is a photograph of the results of the Mycobacterium tuberculosis drug resistance culture test in the comparison kit (arrows indicate Mycobacterium tuberculosis colonies). As shown in FIG. 4, in the kit using the conventional culture composition, the color of the Mycobacterium tuberculosis colony was similar to the medium composition, and the boundaries of the Mycobacterium tuberculosis colony were vague, so it was not easy to count the colonies, so it was not easy to determine drug resistance.

5A to 5E are photographs showing a case in which drug resistance is not confirmed as a result of a Mycobacterium tuberculosis drug resistance culture test using the 5 kits of I to V according to the present invention. In these kits, Mycobacterium tuberculosis colonies were detected only in the positive control group. It is confirmed that Mycobacterium tuberculosis proliferated well without inhibition of proliferation in the positive control medium of all five kits of the present invention, and it can be seen that the medium composition according to the present invention enables the detection of Mycobacterium tuberculosis without inhibiting the proliferation of Mycobacterium tuberculosis. have.

6A to 6E are photographs showing a case in which drug resistance is detected as a result of a Mycobacterium tuberculosis drug resistance culture test using the 5 kits of I to V according to the present invention.

Figure 6a is the test result using the kit I of the present invention. As shown in Figure 6a, the first patient (I-2-1) and the second patient (I-2-2) are group 1 antituberculosis drugs. It could be easily found that all of the four oral first-line anti-tuberculosis antibiotics were resistant.

Figure 6b is the test result using the kit II of the present invention. As shown in Figure 6b, both patients (II-2-1, II-2-2) are among the second anti-tuberculosis agents for injection, which are group 2 anti-tuberculosis agents. It could easily be found that both were resistant to streptomycin.

Figure 6c is the test result using the kit III of the present invention. As shown in Figure 6c, both patients (III-2-1, III-2-2) are quinoline antibiotics (levofloxacin and Moxifloxacin) and high-dose streptomycin could easily be found to be resistant.

6D is the test result using the kit IV of the present invention. As shown in FIG. 6D , the first patient (IV-2-1) was a group 4 antituberculosis, an oral secondary antituberculosis agent, among three antibiotics, cyclo Resistance to serine could be easily proved, and the second patient (IV-2-2) was easily found to be resistant to cycloserine and P-aminosalicylic acid among three antibiotics, which are oral second-line antituberculosis drugs. can do.

Figure 6e is the test result using the kit V of the present invention. As shown in Figure 6e, both patients (V-2-1, V-2-2) are Della out of three new anti-tuberculosis drugs in group 5. It could be easily proved that he was resistant to manid.

1A and 1B are photographs showing the proliferated Mycobacterium tuberculosis colonies and their counting as a result of testing using the kit I of the present invention. Arrows are indicated to count Mycobacterium tuberculosis colonies, and a total of 50 colonies were counted with 10 yellow, 10 green, 10 light purple, 10 deep yellow, 7 purple, and 3 red.

Summarizing the above results, it can be seen that, when the kit according to the present invention is used, the Mycobacterium tuberculosis is proliferated well and it is easy to detect the proliferated Mycobacterium tuberculosis colony, so it can be seen that the Mycobacterium tuberculosis drug resistance detection can be easily and accurately performed. In addition, when the five kits according to the present invention are used in an appropriate combination for each tuberculosis patient, it is possible to easily detect Mycobacterium tuberculosis drug resistance in a short time and to prescribe a patient-specific anti-tuberculosis agent.

Claims (9)

A medium composition for detecting drug-resistant Mycobacterium tuberculosis comprising food color blue No. 1 as an active ingredient,
The food color blue No. 1 is 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3-sulfonatebenzyl)amino]phenyl](2-sulfonatephenyl)methylene ]-2,5-cyclohexadienylidene]ammoniomethyl]benzenesulfonic acid disodium contains 85.0% (w/w) or more,
The composition is a medium composition comprising food color blue No. 1 in an amount of 0.01 to 0.03 g/L.
delete delete The medium composition for detecting drug-resistant Mycobacterium tuberculosis according to claim 1, wherein the medium composition comprises Middlebrook 7H10 agar medium and Middlebrook OADC growth supplement.
The medium composition according to claim 1; and
A kit for detecting drug-resistant Mycobacterium tuberculosis including a case,
The case is a kit for detecting drug-resistant Mycobacterium tuberculosis having a structure in which 6-wells having a diameter of 35 mm are mounted on a fixed plate.
The kit for detecting drug-resistant Mycobacterium tuberculosis according to claim 5, wherein the 6-well contains two antibiotic-free medium compositions and up to four antibiotic-added medium compositions.
[Claim 7] The kit for detecting drug-resistant Mycobacterium tuberculosis according to claim 6, comprising an antibiotic-added medium composition to which an antibiotic is added as any one selected from the following groups:
i) Ethambutol 5.0 ug/ml, isoniazid 0.2 ug/ml, rifampicin 1.0 ug/ml, and rifabutin 0.5 ug/ml are added to four antibiotic-added medium compositions, respectively;
ii) four antibiotic-added medium compositions each containing amikacin 4.0 ug/ml, kanamycin 5.0 ug/ml, capreomycin 10.0 ug/ml, and streptomycin 2.0 ug/ml;
iii) levofloxacin 1.0 ug/ml, moxifloxacin 0.5 ug/ml, isoniazid 1.0 ug/ml, streptomycin 10.0 ug/ml 4 antibiotic-added medium compositions each added;
iv) Prothionamide 5.0 ug/ml, cycloserine 30.0 ug/ml, and pars (P-aminosalicylic acid) 2.0 ug/ml are added to three antibiotic-added medium compositions, and
v) Three antibiotic-added medium compositions each containing 0.25 ug/ml of bedaquiylin, 1.0 ug/ml of linezolid, and 0.016 ug/ml of delamanide.
i) collecting a sample of sputum from an individual and culturing enrichment; and
ii) A method for detecting drug-resistant Mycobacterium tuberculosis comprising the step of inoculating and culturing an enriched culture medium in the kit according to any one of claims 5 to 7.
The method for detecting drug-resistant Mycobacterium tuberculosis according to claim 8, wherein the subject is a tuberculosis patient or a human suspected of tuberculosis.

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