WO2003031654A1

WO2003031654A1 - Microarray comprising probes for mycobacteria species genotyping, m. tuberculosis strain differenciation, and antibiotic-resistant strain detection

Info

Publication number: WO2003031654A1
Application number: PCT/KR2002/001885
Authority: WO
Inventors: Cheol-Min Kim; Hee-Kyung Park; Hyun-Jung Jang; Eun-Sil Song
Original assignee: Sj Hightech Co.,Ltd.
Priority date: 2001-10-09
Filing date: 2002-10-09
Publication date: 2003-04-17
Also published as: KR20030030266A; KR100454585B1

Abstract

A microarray having probes for genotyping Mycobacteria species, differentiating Mycobacterium tuberculosis strains, and detecting antibiotic-resistant strains, immobilized on the support, a method for genotyping Mycobacteria species, differentiating Mycobacterium tuberculosis strains, and detecting antibiotic-resistant strains using the microarray, and a diagnostic kit for tuberculosis and non-tuberculosis Mycobacteria infections using the same are provided. The microarray includes Mycobacteria species genotyping probes, Mycobacterium tuberculosis strains differentiating probes, and antibiotic-resistant strains detecting probes together. When a plurality of those probe sets are immobilized on a single support, a Mycobacterium genotyping test, a M. tuberculosis strain differentiation test, and an antibiotic-resistance detection test can be simultaneously performed on multiple clinical isolates through a single test, thereby reducing time consumption and costs. Clinical isolates as well as strain cultures can be directly applied to the microarray so that the time for diagnosis is further reduced. Also, the test results are accurate.

Description

MICROARRAY COMPRISING PROBES FOR MYCOBACTERIA

SPECIES GENOTYPING, M. tuberculosis STRAIN DIFFERENCIATION,

AND ANTIBIOTIC-RESISTANT STRAIN DETECTION

Technical Field

The present invention relates to a method for simultaneously genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains, and more particularly, to a microarray comprising on a support probes for genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains, a detection method using the microarray, and a diagnostic kit using the microarray. Background Art

Mycobacteria are major pathogens causing human diseases. In the world, eight million people are infected every year by Mycobacterium tuberculosis and three million people among them die from the infection (Raviglione, M. O, D. E. Snider, and A. Kochi, "Global epidemiology of tuberculosis, morbidity and mortality of a worldwide epidemic," JAMA, 271 :220-226, 1995). Recently, as the number of peoples suffering from AIDS rapidly increases, infections caused by non-tuberculosis mycobacteria (NTM) are gradually increasing (Barnes, P., A. B. Bloch, P. T. Davidson, and D. E. Snider, "Jr. Tuberculosis in patients with immunodeficiency virus infection," N. Engl. J. Med., 324:1644-1650, 1991 ). For this reason, there is an urgent need to develop a method for rapidly and efficiently identifying non-tuberculosis mycobacteria as well as tuberculosis mycobacteria and diagnosing infections caused by the same.

Most conventional methods for identifying and classifying bacteria are based on the morphological, biochemical, and growth characteristics of bacteria. These conventional methods are very complicated to conduct and take much time. Tuberculosis surveillance involves asking a patient detailed questions on his/her condition, clinical tests, chest X-rays, tuberculin tests, blood tests, and bacteriological tests. Conventionally, tuberculosis strains in a patient's sputum were microscopically observed after smearing and staining or after proliferation in test tubes. However, a small number of tuberculosis strains in a sample cannot be detected by the smearing and staining method. The cultivation method takes much time, about 6 weeks, since the growth of tuberculosis strains is very slow. Due to these problems, use of simple and rapid identification methods using target gene sequences have become more common. For emerging gene-based identification methods, genus-specific or species-specific PCR primers or nucleotide probes are applied to a target gene of interest.

Under this situation, pedigree analysis on Mycobacteria, which would provide the base for species identification, has been conducted by comparison of 16S rRNA or its nucleotide sequences. This is based on the fact that the16S rRNA gene has the conserved but polymorphic sequences for Mycobacteria species identification (Stahl, D. A., and J. W. Urbane, "The division between fast and slow-growing species corresponds to natural relationships among the Mycobacteria," J. Bacteriol., 172:116-124 (1990); Rogall, T., J. Wolters, T. Flohr, and E. C. Bottger, "Toward a phylogeny and definition of species at the molecular level within the genus Mycobacterium,'' Int. J. Syst. Bacteriol., 40:323-30 (1990b); Rogall T., T. Flohr, and E. C. Bottger, "Differentiation of Mycobacterium species by direct sequencing of amplified DNA," J. Gen. Microbiol. , 136 (Pt9): 1915-1920 ( 1990a)). However, since the 16S rRNA gene has similar nucleotide sequences among some species, there is a limitation in identifying species to a certain extent (Fox, G. E., J. D. and P. J. Jurtshum, "How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity," Int. J. Syst. Bacteriol. 42:166-170 (1992)). Also, IS6110 insertion element has multiple copy numbers in TB complexes of M. tuberculosis, M. africanum, M. bovis, and M. microti, and thus a PCR probe method using IS6110 as a target sequence is used. However, Mycobacterium tuberculosis which do not have the IS6110 insertion element have been reported and thus may yield pseudo-negative test results (Yuen L. K., B. C. Ross, K. M. Jackson, and B. Dwyer, "Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization," J. Clin. Miclobiol., 31:1615-1618 (1993)). As infections caused by non-tuberculosis mycobacteria (NTM), and particularly by unidentified new species of NTM, are increasing, there is a need for the development of a new species-specific gene-based identification and diagnosis method so as to accurately identify disease-causing species to prevent and remedy infections caused by new species of NTM.

When a patient is infected with Mycobacterium tuberculosis, it is important that the source strain that caused the infection be identified and the best treatment be provided according to the type of the identified tuberculosis strain. Conventionally, M. tuberculosis strains, i.e., subspecies, were differentiated by restriction fragment length polymorphism (RFLP) based on the amplification of a tuberculous DNA fragment by polymerase chain reaction (PCR). In the RFLP technique, a site of the amplified IS6110 fragment that can be recognized by a restriction enzyme is utilized. However, since this DNA RFLP technique uses DNA from strain cultures, it is not suitable for slow-glowing strains. Furthermore, it needs elaborate instrumental handling (Goyal M., Saunders N.A., van Emdden J.D., Young D.B., Shaw R.J. Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism. J. Clin Microbiol, 35:647-651 (1997)). Alternatively, TB complexes can be identified using DNA fingerprinting by southern blotting of genomic DNAs or using polymorphic GC-rich sequences (PGFS) or repetitive elements such as a triplet multimer (GTG). However, due to the target DNA polymorphism with respect to primers for PCR amplification and the diverse sizes of the target DNAs, those methods cannot be applied to simultaneous extraction and differentiation of the strains in a number of clinical isolates_(Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, and van Embden J. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology, J. Clin Microbiol, 35:907-14 (1997)).

Currently, a miniblotter for differentiating M. tuberculosis strains using non-repetitive spacers of different lengths present within the direct repeat (DR) locus is commercially available (MiniSlot Apparatus from Immunetics Inc., USA). However, it takes about 2-3 days to obtain results because of the multiple processes involved, such as hybridization and detection (van Embden J.D., van Gorkom T., Kremer K., Jansen R., van Der Zeijst. B.A., and Schouls L.M. Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria., J. Bacteriol, 182L2393-401 (2000); Filliol I., Sola C, and Rastogi N., Detection of a previously unamplified spacer within the DR locus of Mycobacterium tuberculosis: epidemiological implications., J. Clin. Microbiolol, 38:1231-1234 (2000)). Multidrug-resistant tuberculosis due to the misuse or abuse of antibiotics has become a serious concern worldwide. In impoverished countries, the number of individuals with continued latent infection with antibiotic-resistant tuberculosis strains increased as a result of improper medical treatment due to a shortage of anti-tuberculosis drugs. In a recent survey of antibiotic-resistant tuberculosis strains conducted in 35 countries over 5 years, it was found that 13% of the cases were resistant to at least one antibiotic, and about 7.5% had multidrug-resistant M. tuberculosis (MDR-TB), which is more serious because the strains are resistant to two or more kinds of antibiotics, which include isoniazid (INH) and rifampin (RMP). For antibiotic-resistant tuberculosis, among other tuberculosis cases, concerns for a healthy life of the infected individual are more serious. Therefore, there is a need for early detection of the MDR-TBs for the effective treatment of patients and to suppress the incidence of MDR strains and for the development of a rapid antibiotic susceptibility measurement method. In most clinical laboratories, a standard tuberculosis detection in which an acid-fast bacteria smear or acid-fast bacteria staining using tissue is followed by biochemical culture confirmation is used as an antibiotic susceptibility test for tuberculosis strains (Kent B.D., Kubica G.P. Public health mycobacteroiology; a guide for the level III laboratory, US Department of Health and Human Services, Atlanta: Centers for Disease Control, 207(1985)). It takes 4-6 weeks from sampling to the identification of tuberculosis strains. Since the strains are cultured in Middlebrook media, it takes 8-12 weeks for the antibiotic susceptibility test. Therefore, there is a limit to how quickly the antibiotic-resistant strains can be detected.

Owing to the active molecular biological research on the mechanism of antibiotic resistance, a variety of genetic variations which induces resistance to rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB), streptomycin (STR), fluoroquinolone (FZ), kanamycin, and amikacin were disclosed. However, a method for simultaneously detecting various mutations using the genetic variations of antibiotic resistance has not yet been reported.

As described above, there have been many cases of infections by Mycrobacteria and death from the infections worldwide. Recently, non-tuberculosis infections as well as tuberculosis infections are gradually increasing due to the abrupt worldwide spread of acquired immunodeficiency syndrome (AIDS). In addition, the incidence of MDR-TBs due to the misuse or abuse of antibiotics is also serous. Therefore, rapid, efficient detection and identification of tuberculosis and non-tuberculosis species, epidemiological studies on the tuberculosis species, and detection of antibiotic-resistant tuberculosis strains are needed. In the current situation, Mybocabtaria species genotyping, M. tuberculosis strain differentiation, and determination of antibiotic resistance of the tuberculosis strains are separately performed, which takes a longer period of time and delays treatment.

Disclosure of the Invention

As the result of efforts made to improve drawbacks and inefficiency in the conventional biochemical identification methods accompanying strain cultivation and thus taking at least 6-weeks or longer, the inventors have developed a microarray comprising tuberculosis and non-tuberculosis Mycobacteria species-specific probes, M. tuberculosis strain-specific probes, and antibiotic-resistant strain-specific probes and have found that tuberculosis and non-tuberculosis Mycobacteria species genotyping, M. tuberculosis strain differentiation, and antibiotic-resistant strain detection can be accurately and quickly performed on clinical isolates as well as strain cultures through a single test using the microarray according to the present invention.

The present invention provides a microarray capable of accurately and rapidly genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains through a single test.

The present invention also provides a method for detecting tuberculosis and non-tuberculosis Mycobacteria strains and a diagnostic kit for tuberculosis and non-tuberculosis Mycobacteria infections using the microarray. The present invention also provides a probe or primer oligonucleotide for use in the microarray.

In one aspect of the present invention, there is provided a microarray comprising: a support; a first probe for genotyping Mycobacteria species, immobilized on the support; a second probe for differentiating Mycobacteria tuberculosis strains, immobilized on the support; and a third probe for detecting antibiotic-resistant strains, immobilized on the support.

In the microarray according to the present invention, suitable supports include, but not limited to, a glass slide, a membrane, a semi-conductive chip, a silicon substrate, etc.

It is preferable that a plurality of probe sets, each of the probe sets comprising the first probe for genotyping Mycobacteria species, the second probe for differentiating Mycobacterium tuberculosis strains, and the third probe for detecting antibiotic-resistant strains, are immobilized on the single support. In this case, genotyping Mycobacteria species, differentiating Mycobacterium tuberculosis strains, and detecting antibiotic-resistant strains can be simultaneously performed using multiple samples through a single test within a short period of time. Also, the results are accurate.

In the microarray according to the present invention, the first probe for genotyping Mycobacteria species includes any oligonucleotide having a sequence that can specifically bind to a target gene of tuberculosis and non-tuberculosis Mycobacterium species, for example, the known target nucleotide sequence of the 16S rRNA, IS6110 genes, the HSP gene, or the SOD gene. Preferably, the first probe for genotyping Mycobacteria species includes a Mycobacterium genus-specific oligonucleotide based on the ITS (internal transcribed spacer) sequence, an oligonucleotide having the Mycobacterium tuberculosis species-specific ITS sequence, and an oligonucleotide having the Mycobacterium non-tuberculosis species-specific ITS sequence. More preferably, the first probe for genotyping Mycobacteria species includes at least one of the oligonucleotides of SEQ ID NOs. 1 through 9, at least one of the oligonucleotides of SEQ ID NOs. 10 through 16, and at least one of the oligonucleotides of SEQ ID NOs. 17 through 89.

The ITS region used as a target gene for genotyping Mycobacteria species in the present invention is present between the 16S rRNA and 23S rRNA genes of the Mycobacteria and has a different size of about 300-500 bp according to the types of the strains. The ITS region has the Mycobacterium genus-specific conserved sequences and species-specific sequences, and thus is preferred as a target nucleotide sequence for identification primers (probes).

A microarray for genotyping Mycobacteria species according to the present invention is manufactured by attaching the Mycobacterium genus-specific and species-specific probes prepared using the highly polymorphic ITS region as the target nucleotide sequence to the support. A pair of primers is derived from the conserved sequences at both sides of the ITS region and amplified by polymerase chain reaction (PCR). The hybridization of the amplified sample to the probes on the microarray is detected as a stain identification signal. The microarray according to the present invention can diagnose double infections by multiple strains through a single test, which could not be detected by conventional methods.

In the microarray according to the present invention, the second probe for differentiating M. tuberculosis strains includes any oligonucleotide having a sequence that can specifically bind to a target gene of M. tuberculosis strains, for example, a target nucleotide sequence of the IS6110 gene, a repetitive polymorphic GC-rich sequence (PGRS), or a triplet multimer (GTG) sequence, which are already known. Preferably, the second probe for differentiating M. tuberculosis strains includes an oligonucleotide of the direct repeat (DR) sequence specific to M. tuberculosis strains. More preferably, the second probe for differentiating M. tuberculosis strains includes at least one of the oligonucleotides of SEQ ID NOs. 90 through 134.

According to the present invention, M. tuberculosis strains are differentiated by a microarray manufactured using the DR locus of M. tuberculosis or non-repetitive spacers of different lengths within the DR locus as a target probe, based on the polymorphism of the DR locus in different tuberculosis strains, as in spoligotyping. The differentiation of M. tuberculosis strains in the present invention is based on the hybridization of the M. tuberculosis strains amplified through in vitro PCR to the target probe. This method for differentiating M. tuberculosis strains is more accurate, rapid, and convenient than conventional commercialized methods.

In the microarray according to the present invention, the third probe for detecting antibiotic-resistant strains includes any oligonucleotide having a sequence that can specifically bind to a target gene inducing antibiotic resistance, for example, a target nucleotide sequence inducing resistance to rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB), streptomycin (STR), fluoroquinolone (FQ), kanamycin, amikacin, etc. More preferably, the third probe for detecting antibiotic-resistant strains includes an oligonucleotide having a point mutation sequence of the rpoB gene that induces resistance to RMP, an oligonucleotide having a point mutation sequence of the katG gene that induces resistance to INH, and an oligonucleotide having a point mutation sequence of the pncA gene that induces resistance to PZA. More preferably, the third probe for detecting antibiotic-resistant strains includes at least one of the oligonucleotides of SEQ ID NOs. 135 through 160, at least one of the oligonucleotides of SEQ ID NOs. 161 through 169, and at least one of the oligonucleotides of SEQ ID NOs. 170 through 173.

Among multidrug-resistant tuberculosis strains resistant to both RMP and INH, 94-98% of RMP-resistant strains has a point mutation in the restricted j3 subunit region of DNA-dependent RNA polymerase which is encoded by the rpoB gene. Accordingly, the RMP-resistant strains can be easily identified using hybridization or reverse hybridization of the DNA.

INH as an antibiotic widely used in treating infections by M. tuberculosis complex has resistance in non-tuberculosis Mycobacteria and prokarytes. About 84% of INH-resistant tuberculosis strains has a mutation in the katG gene or an upstream nucleotide substitution in the inhA gene. About 75% of the INH-resistant tuberculosis strains is caused by partial structural changes in the katG gene, and thus functional deterioration or loss occurs in /cafG-encoded catalase-peroxidase. The mechanism of resistance to INH can be explained by mutations with the substitution of threonine (ACC) and asparagine (AAC) for serine (AGC) at codon 315 of the katG gene and the substitution of leucine for arginine (CGG) at codon 463.

In multidrug-resistance tuberculosis, it is more crucial to detect resistance to PZA than to other antibiotics such as INH and RMP because it is difficult to treat PZA-resistant tuberculosis. PZA as the primary anti-tuberculosis drug shows antibacterial activity when converted into pyraxionic acid (POA) by pyrazinamidase (PZase) in tuberculosis strains. However, when the activity of the PZase is lost in a tuberculosis strain, the tuberculosis strain becomes resistant to PZA.

According to the present invention, a method for detecting antibiotic-resistant strains is based on molecular genetics, and particularly, the detection of mutations caused by changes in the nucleotide sequence associated with the activity of antibiotics. A microarray manufactured by immobilizing probes for detecting wide-type and mutant-type target sequence of the antibiotic resistant gene on a solid support is used. Different kinds of mutations can be accurately detected through a single test within a short period of time, compared to conventional time-consuming antibiotic-resistant tuberculosis strain detecting methods, in order to timely, effectively treat the infections.

In another aspect, the present invention provides a method for simultaneously genotyping Mycobacteria species, differentiating Mycobacterium tuberculosis strains, and detecting antibiotic-resistant strains using the microarray according to the present invention as described above. In particular, after manufacturing the microarray including those probes according to the present invention, a target DNA isolated from strain cultures or clinical samples is hybridized to the probes, and it is detected whether or not the target DNA is bound to the probes. As a result, Mycobacteria species genotyping, Mycobacterium tuberculosis strain differentiation, and antibiotic-resistant strain detection can be simultaneously performed within a short period of time. In still another aspect, the present invention provides a diagnostic kit for tuberculosis and non-tuberculosis Mycobacteria infections, comprising the microarray according to the present invention as described above. The diagnostic kit according to the present invention may further include a hybridizing reaction solution, a PCR kit including probes for amplifying the target gene, an unhybridized-DNA washing solution, a cover slip, a dye, a dye-uncoupled product washing solution, a user's manual, etc.

In another aspect, the present invention provides a primer or probe oligonucleotide for genotyping Mycobacteria species, having at least one sequence of SEQ ID NOs. 1 through 89. The present invention also provides a primer or probe oligonucleotide for differentiating Mycobacterium tuberculosis strains, having at least one sequence of SEQ ID NOs. 90 through 134, and a primer or probe oligonucleotide for detecting antibiotic-resistant strains, having at least one sequence of SEQ ID NOs. 135 through 173.

The oligonucleotides for use as the probes to be attached to the microarray for Myctobacteria species differentiation and antibiotic-resistant mutant detection according to the present invention can be used as primers for PCR amplification of a Mycobacterium genus-specific and species-specific target DNA. The oligonucleotides for use as the probes or primers according to the present invention have a sense or antisense sequence of the above-listed sequence ID numbers.

FIG. 1 shows embodiments of a microarray according to the present invention. In FIG. 1, a) shows a microarray including a plurality of probe sets attached to a single support, wherein each of the probe sets includes the first probes for genotyping Mycobacteria species, the second probes for differentiating M. tuberculosis strains, and the third probes for detecting antibiotic-resistant strains. In FIG. 1 , b) shows a microarray including one probe set from a), in which the individual probes for genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains in numbered multiple spots of the support are shown, and c) shows the microarray of b) which is sectioned according to the functions of the probes, wherein the first probes for genotyping Mycobacteria species are attached to the upper left area of the microarray, the second probes for differentiating M. tuberculosis strains are attached to the upper right area, and RMP-resistant strain detecting probes among the third probes are attached to the middle left area, INH-resistant strain detecting probes are attached to the lower left area, and PZA-resistant strain detecting probes are attached to the lower right area. The layout of the microarray according to the present invention in c) is for illustrative purposes, and thus it will be appreciated that the layout of the microarray for different probes can be varied.

The probes or primers used in the present invention for genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains are shown in Tables 1 , 2, and 3 below, respectively.

Table 1. Primers and probes for genotyping Mycobacteria specis j Mycobacterium species Probe Nucleotide sequence _N0

ITSF

Mycobacteria CGAAGCCAGTGGCCTAACCC 1

ITSR GGATCCTGCCAAGGCATCCACCA 2 YC-01 TGGTGGGGTGTGGTGTΠΓGA 3 MYC-02 TGGATAGTGGTTGCGAGCAT 4 MYC-03 TGGATAGTGAΠGCGAGCAT 5 YC-04 TGGATAGTGCTTGCGAGCAT 6 MYC-05 TGGATAGTGGΠGAGAGCAT 7 MYC-06 TGGATAGTGGΠGGGAGCAT 8 MYC-07 ATAGTGATTGCGAGC 9

TB COMPLEX MTB-01 CACTCGGACTTGTTCCAGGT 10

MTB-02 TGGTGGGGCGTAGGCCGTGA 11

MTB-03 CAACAAAGTTGGCCA 12

MTB-04 GGACTTGTTCCAGGT 13

MTB-05 AGGTGTTGTCCCACC 14

MTB-06 TGCATGACAACAAAG 15

MTB-07 AGGTGTTZTCCCACC 16 aviυm - M. inlracellular MAC-01 CCCTGAGACAACACTCGGTC 17 (MAC) MAC-02 GCGTTCATCGAAATGTGTAAT 18 MAC-03 CTCGGTCGAACCGTG 19

M. fortultum FOR-01 CCGTGAG6AACCGGTTGCCT 20 FOR-02 TAGCACGCAGAATCGTGTGG 21 FOR-03 GTAGT6GGCACGGTTTGGTG 22 FOR-04 CAMCTTTTTTGACTGCCAG 23 FOR-05 AGGCCCGTGCCCCTTTTGGG 24 FOR-06 TGGCATCCGGTTGC6GGTGT 25 FOR-07 GGTTTTGTGTGTTGATGTGC 26

M. chelonaβ CHE-01 GTGGTTACTCGCTTGGT 27 CHE-02 TTGGGAACATAAAGCGAGπ 28 CHE-03 CAATAGAAπGAAACGCTGGCA 29 CHE-04 GTAGTCGGCAAAACGTC6GA 30

M. abscβssus ABC-01 TAAAGTAG6CATCTG 31

ABC-02 GGATATCTACTTGGT 32

ABC-03 TAAACATAGCCTCGCTCGπ 33

M. Qor onae GOR-01 • CGACAACAAGCTAAGCCAGA 34

GOR-02 AAAATGTATGCGTTG 35

G0R-03 TGTCGπCGCGGCAACGT 36

GOR-04 CACCCTCGGGT6CTGTC 37

M. kansasli KAN-01 GCGCAACTGTAAATGAATCA 38

KAN-02 CTGGATGCGCTGCCGπCG 39

KAN-03 MCTGTAAATGAATCACCAACAC 40

KAN-04^' GGACGAAAGCCGGGTGCAC 4.

KAN-05 GCATCCCAACAAGTGGG 42

KAN-06 CTCGGGCTCTGTTCGAG 43

. M: scrofulaceum SCC-01 TCGGCTCGTTCTGAGTGGTG 44 SCO-02 TAAACGGATGCGTGGCCGAA 45

M. szulgal SZU-01 AACACTCAGGCΓΓGGCCAGA 46 SZU-02 CAAΠGGATGCGCTGCCCTC 47 S2U-03 GCGCGGCAACGAACAAGCCA 48 SZU-04 AGGCTTGGCCAGAGCTGTTG 49

M. vaccaβ VAC-01 CGATTCGTTGGATGGCCTTT 50 VAC-02 AATGCCGGCGAGGGAAAT 51 VAC-03 GAATGCACAGCGCTTGTGGT 52 Table 1 (continued)

Table 2. Primers and probes for differentiating M. tuberculosis strains

Table 3. Primers and probes for detecting antibiotic-resistant strains

Brief Description of the Drawings

FIG. 1 shows embodiments of a microarray according to the present invention, in which a) shows a microarray including a plurality of probe sets attached to a single support, each of the probe sets including Mycobacteria species genotyping probes, M. tuberculosis strain differentiating probes, and antibiotic-resistant strain detecting probes, b) shows a microarray including one probe set from a), and c) shows the microarray of b) which is sectioned according to the functions of the probes;

FIG. 2 shows the result of hybridization to the probes for simultaneously genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains through a single test; FIGS. 3A and 3B show the results of hybridization of M. tuberculosis H37Rv strain and non-tuberculosis strain M. scrofulaceum, respectively, to the probes for genotyping Mycobacteria species;

FIG. 4A and 4B show the results of hybridization of M. tuberculosis H37Rv strain and M. bovis, respectively, to the probes for differentiating M. tuberculosis strains; and

FIGS. 5A and 5F show the results of hybridization specific to the probes for detecting the resistance of Mycobacteria to rifampin (RMP), isoniazid (INH), and pyrazinamide (PZA), and particularly, FIGS. 5A and 5B show the results of a PMP resistance test using RMP-susceptible M. tuberculosis H37Rv strain and a RMP-resistant M. tuberculosis strain, respectively, FIGS. 5C and 5D show the results of an INH resistance test using INH-susceptible M. tuberculosis H37Rv strain and an INH-resistant M. tuberculosis strain, respectively, and FIGS. 5E and 5F show the results of a PZA resistance test using PZA-susceptible M. tuberculosis H37Rv strain and a PZA-resistant M. tuberculosis strain, respectively.

Best mode for carrying out the Invention

Hereinafter, embodiments of the present invention will be described in detail with reference to the appended drawings. The present invention is not restricted to the following embodiments, and many variations are possible within the spirit and scope of the present invention. The embodiments of the present invention are provided in order to more completely explain the present invention to one of ordinary skill in the art.

Example 1 : Incubation of Mycobacteria strains and isolation of genomic DNA

Standard strains of Mycobacteria were obtained from the Korean Collection for Type Culture (KCTC) and the American Type Culture Collection (ATCC). Clinical isolates were obtained from the Korean National Tuberculosis Association and National Masan Hospital. The DNA in the strains and clinical isolates were extracted using InstaGene matrix (Bio-Rad Co. USA).

To extract DNAs from the strains and clinical isolates, 200 μL InstaGene matrix was put into a 1.5 mL tube. A target strain was incubated in a solid medium (Ogawa plate), one scrapping of the strain layer was removed with an inoculating loop and suspended in the InstaGene Matrix contained in the tube. The suspension was reacted at 56°C for 30 minutes and mixed thoroughly for 10 seconds. The mixture was heated at 100°C for 8 minutes and mixed thoroughly for 10 seconds. The mixture was centrifuged at 12,000 rpm for 3 minutes and the supernatant was collected as a template DNA for PCR.

The standard strains used were:

M. tuberculosis H37Rv (ATCC 27294) M. fortuitum (ATCC 6841)

M. flavescens (ATCC 14474)

M. avium (ATCC 25291)

M. intracellulare (ATCC 13950)

M. kansasii (ATCC 12478) M. chelonae (ATCC 35752) M. abscessus (ATCC 19977) M. szulgai (ATCC 35799) M. scrofulaceum (ATCC 19981 ) M. gordonae (ATCC 14470) M. vaccae (ATCC 15483) M. xenopi (ATCC 19250) M. smegmatic (ATCC 21701 ) M. genavense (ATCC 51233) M. malmoense (ATCC 29571) M. simiae (ATCC 25275) M. marinum (ATCC 927) M. ulcerans (ATCC 19423) M. gastri (ATCC 15754) M. terrae (ATCC 15755)

M. leprae clinical isolates

M. tuberculosis clinical isolates

M. bovis BCG F117392

Example 2: Preparation of probes for genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting of antibiotic-resistant strains

To genotype Mycobacteria species, differentiate M. tuberculosis strains, and detect antibiotic-resistant strains, gene-specific oligonucleotide probes each including 15-25 nucleotides with a dT spacer of a length of 15 nucleotides at 5'- end were synthesized using a DNA synthesizer (Perkin Elmer, USA) and isolated by PAGE. The nucleotide sequences of the primers and probes for genotyping of Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains are shown in Tables 1 , 2, and 3 above, respectively. However, any primer and probe having, but not limited to, the sequences in Tables 1 , 2, and 3 can be used for those purposes of the present invention.

1. Preparation of the first probes for Mycrobacteria species genotyping 1 ) Preparation of Mycobacteria genus-specific probes

For hybridization only with the species within the genus Mycobacterium, Mycobacteria genus-specific internal transcribed spacer (ITS) regions having conserved sequences, which selectively hybrid only to Mycobacteria species, not to other pathogenic microorganisms, were selected. The probes, MYC-01 (SEQ ID NO. 1) through MYC-07 (SEQ ID NO. 9) in Table 1 , were prepared using the selected sequences.

2) Preparation of M. tuberculosis species-specific probes Probes that specifically hybrid only to M. tuberculosis complex species, not to the other species within the genus Mycobacterium, were prepared using the ITS sequences of the M. tuberculosis complex and non-tuberculosis Mycobacteria. The probes include MTB-01 (SEQ ID NO. 10), MTB-02 (SEQ ID NO. 11), MTB-03 (SEQ ID NO. 12), MTB-04 (SEQ ID NO. 13), MTB-05 (SEQ ID NO. 14), MTB-06 (SEQ ID NO. 15), and MTB-07 (SEQ ID NO. 16).

3) Preparation of non-tuberculosis species-specific probes Probes that specifically hybrid only to non-tuberculosis

Mycobacteria species were prepared using the species-specific ITS sequences of the non-tuberculosis Mycobacteria. A total of 73 probes including from MAC-01 (SEQ ID NO. 17) to LEP-04 (SEQ ID NO. 89) in Table 1 , capable of differentiating 21 kinds of non-tuberculosis Mycobacteria species, were obtained.

2. Preparation of the second probes for M. tuberculosis strain differentiation A total of 43 probes, including from SPO-01 (SEQ ID NO. 92) to SPO-43 (SEQ ID NO. 134) in Table 2, which are specific to tuberculosis strains, were prepared using the direct repeat (DR) locus of the M. tuberculosis and non-repetitive spacers having diverse lengths of 35-41 bp present within the DR locus. The 43 sequences for M. tuberculosis strain differentiation are described in various publications (van Embden J.D., van Gorkom T., Kremer K., Jansen R., van Der Zeijst B.A., Schouls L.M., Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria, J. Bacteriol., 182:2393-2401 (2000); Kamerbeek J., Schouls L, Kolk A., van Agterveld M., van Soolingen D., Kuijper S., Bunschoten A., Molhuizen H., Shaw R., Goyal M., van Embden J., Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology, J. Clin. Microbiol., 35:907-914 (1997)).

3. Preparation of the third probes for antibiotic-resistant strain detection

1 ) Preparation of probes for detecting rifampin-resistant M. tuberculosis strains For rifampin-resistant M. tuberculosis strain detection, mutation detecting probes having point mutation sequences at the center region were prepared using the sequences of 69 bp from codon 511 to codon 533 of the rpoB gene, where 90% or more of rifampin-resistant M. tuberculosis strains have mutations (Musser J.M., Antimicrobial agent resistance in Mycobacteria: molecular genetic insights. Clin Microbiol. Rev. 8:496-514 (1995); M.K. Lee, Y.S. Kim, H.J. Lee, D.S. Chun, S.M. Yoon, S.S. Park, CM. Kim, and S.K. Park, Assay of the rpoB gene of rifampin-resistant tuberculosis strains by sequencing and a line probe assay, Tuberculosis & Respiratory Disease, 44:251-263 (1997); Valim A.R., Rossetti M.L., Ribeiro M.O., Zaha A., Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from Brazil., J. Clin. Microbiol., 38: 3119-3122 (2000)). Point mutation sequences were used, in which leucine (CTG) at codon 511 was replaced by proline (CCG), glutmine (CAA) at codon 513 was replaced by leucine (CTA), aspartic acid (GAC) at codon 516 was replaced by valine (GTC) and tyrosine (TAC), serine (TCG) at codon 522 was replaced by leucine (TTG), histidine (CAC) at codon 526 was replaced by tyrosine (TAC), aspartic acid (GAC), arginine (CGC), leucine (CTC), and proline (CCC), serine (TCG) at codon 531 having the highest incidence of mutations was replaced by leucine (TTG) and tryptophan (TGG), and leucine (CTG) at codon 533 was replaced by proline (CCG). A total of 24 probes, from SEQ ID NO. 137 to 160 in Table 3, capable of detecting 7 kinds of wide-type M. tuberculosis and 13 kinds of its mutant strains, were prepared.

2) Preparation of probes for detecting isoniazid-resistant M. tuberculosis strains

For isoniazid-resistant M. tuberculosis strain detection, mutation detection probes having point mutation sequences at the center region were prepared using the sequences at codon 315 and codon 463 of the katG gene, where 70% or more isoniazid-resistant M. tuberculosis strains have mutations (Musser J.M., Kapur V., Williams D.L., Kreiswirth B.N., van Soolingen D., van Embden J.D., Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance., J. Infect. Dis., 183:196-202 (1996)). Point mutation sequences were used, in which serine (AGC) at codon 315 was replaced by threonine (ACC) and asparagine (AAC), and arginine (CGG) at codon 463 was replaced by leucine (CTG). The probes from SEQ ID No. 165 to SEQ. ID No. 169 in Table 3 were prepared to detect 2 kinds of wide-type M. tuberculosis and 3 kinds of its mutant strains.

3) Preparation of probes for detecting pyrazinamide-resistant M. tuberculosis strains For pyrazinamide-resistant M. tuberculosis strains, mutations scatter over the pncA gene sequence. Probes were designed to enable detection of a point mutation, the substitution of 'G' for 'A' at -11 site of the promoter where the incidence of mutations is relatively high. As a result, the primer (SEQ ID. NO 172) and the probe (SEQ ID NO. 173) in Table 3 were obtained to detect one kind of wide-type M. tuberculosis and one kind of its mutant strain.

Example 3: Preparation of target DNA

1. Preparation of target DNA for Mycobacteria species genotyping

To amplify a target DNA for genotyping Mycobacteria species, primers capable of selectively amplifying the ITS region including parts of the 16S rRNA and 23S rRNA to 300-500 bp were prepared using biotin-labeled ITSF 5'-biotin-CGA AGC CAG TGG CCT AAC CC-3' (SEQ ID NO. 1), MYC-02 5'-biotin-ATG CTC GCA ACC ACT ATC CA-3' (SEQ ID NO. 2), and ITSR 5'-biotin-TGG ATC CTG CCA AGG CAT CCA CCA T-3' (SEQ ID NO. 3) by a common method. The Mycobacteria standard strains and clinical isolates in Example 1 were amplified using the primer by polymerase chain reaction (PCR). PCR was performed using a Perkin-Elmer Cetus Thermocycler Model 9600. After sufficient denaturation at 94°C for 3 minutes, 30 cycles of amplification at 94°C for 1 minute, at 62^*C for 1 minute, and at 72°C for 1 minute were carried out and followed by a single final extension at 72°C for 10 minutes. After the reaction, the PCR products were analyzed by electrophoresis on a 2% agarose gel for molecular weight measurement. This method is described in J. Clin. Microbiol. 38: 4080-4075 (2000) by Park H.K., which is incorporated herein by reference herein.

2. Preparation of target DNA for M. tuberculosis strain differentiation

To amplify a target DNA for differentiating M. tuberculosis strains, a pair of primers, which are disclosed in various articles, capable of amplifying the target DNA to diverse sizes were prepared using biotin-labeled DRa 5'-biotin-GGT TTT GGG TCT GAC GAC-3' (SEQ ID NO. 90) and DRb 5'-biotin-CCG AGA GGG GAC GGA AAC-3' (SEQ ID NO. 91). The Mycobacteria standard strains and clinical isolates in Example 1 were amplified using the primers by PCR. PCR was performed using a Perkin-Elmer Cetus Thermocycler Model 9600. After sufficient denaturation at 96°C for 3 minutes, 25 cycles of amplification at 96°C for 1 minute, at 55°C for 1 minute, and at 72°C for 30 seconds were carried out and followed by a single final extension at 72°C for 10 minutes. After the reaction, the PCR products were analyzed by electrophoresis on a 2% agarose gel in order to identify the PCR products.

3. Preparation of target DNA for antibiotic-resistant M. tuberculosis strain detection

To amplify a target DNA for detecting a mutation in the rpoB gene as a measure of RMP resistance, two pairs of primers capable of amplifying the target DNA to about 160 bp was prepared using biotin-labeled rpoF 5'-biotin-TGC ACG TCG CGG ACC TCC A-3' (SEQ ID NO. 135) and rpoR 5'-biotin-TCG CCG CGA TCA AGG AGT-3' (SEQ ID NO. 136) by a common method. The standard M. tuberculosis H37Rv strain, RMP-resistant M. tuberculosis clinical isolates, and RMP-susceptible M. tuberculosis clinical isolates were amplified using the primer by PCR. PCR was performed using the following temperature profile: sufficient denaturation at 94°C for 3 minutes, 35 cycles of amplification at 94°C for 30 seconds, at 62°C for 30 seconds, and at 72°C for 1 minute, and a single final extension at 72°C for 10 minutes.

To amplify a target DNA for detecting a mutation in the katG gene as a measure of INH resistance, two pairs of primers capable of amplifying the target DNA to 150 bp were prepared. As biotin-labeled primers, katl F 5'-biotin-AAG AGC TCG TAT GGC ACC GG-3' (SEQ ID NO. 161 ) and kat2R 5'-biotin-AGC GCC AGC AGG GCT CTT C-3' (SEQ ID NO. 162) were prepared for PCR of the codon 315 region, and kat3F 5'-biotin-GGC GAA GCC GAG ATT GCC AG-3' (SEQ ID NO. 163) and kat4R 5'-biotin-CTG CAG GCG GAT GCG ACC A-3' (SEQ ID NO. 164) were prepared for PCR of the codon 463 region. The standard M. tuberculosis H37Rv strain, INH-resistant M. tuberculosis clinical isolates, and INH-susceptible M. tuberculosis clinical isolates were amplified using the primers by PCR.

To amplify a target DNA for detecting a mutation in the promoter-11 region of the pncA gene as a measure of PZA resistance, primers capable of amplifying the target DNA to about 200 bp were prepared. As biotin-labeled primers, pncF 5'-biotin-GCT GGT CAT GTT CGC GAT CG-3' (SEQ ID NO. 170) and pncR 5'-biotin-ACC GGT TAC CGC CAG CGA G-3" (SEQ ID NO. 171 ) were prepared. The standard M. tuberculosis H37Rv strain, PZA-resistant M. tuberculosis clinical isolates, and PZA-susceptible M. tuberculosis clinical isolates were amplified using the primers by PCR. PCR was performed using the following temperature profile: sufficient denaturation at 94°C for 3 minutes, 30 cycles of amplification at 94°C for 1 minute, at 62°C for 1 minute, and at 72°C for 1 minute, and a single final extension at 72°C for 10 minutes. After the reaction, the PCR products were analyzed by electrophoresis on a 2% agarose gel. As a result, PCR products of 150 bp and 180 bp were identified.

Example 4: Probe immobilization on support Each of the first, second, and third probes prepared in Example 2 was diluted to a concentration of 100 pmol, transferred into a 96-well microplate, and mixed with a micro-spotting solution to a concentration of 30 pmol. The probes were attached to a support, such as a glass slide or membrane, using a microarrayer (Cartesian Technoloies, PLXSYS 7500 SQXL Microarrayer, USA). One typical probe for each of the Mycobacteria species was selected. In FIG. 1 , the first probes for genotyping Mycobacteria species in spots 1 through 21 correspond to SEQ ID NOs. 4, 13, 17, 25, 27, 31 , 38, 43, 33, 49, 51 , 54, 59, 62, 65, 71 , 75, 77, 81 , 88, and 4 in Table 1 , respectively. The second probes for differentiating M. tuberculosis strains in spots 49 through 93 correspond to a positive control probe, SEQ ID NOs. 92 through 134 in Table 2, and a positive control probe, respectively. Among the third probes for detecting antibiotic-resistant probes, the RMP-resistance detecting probes in spots 22 through 42 correspond to a positive control probe, SEQ ID NOs. 137 through 153, 156, 159, and 160 in Table 3, respectively, the INH-resistance detecting probes in spots 43 through 48 correspond to a positive control probe and SEQ ID NOs. 165 through 169 in Table 3, respectively, and the PZA-resistance detecting probes in spots 94 through 96 correspond to a positive control probe and SEQ ID NOs. 172 and 173 in Table 3, respectively. Two droplets of each of the probes were spotted onto the support. The support with the probe spots was left in a slide box at room temperature for 24 hours or in a dry oven at 50°C for about 5 hours to fix the probes to the surface of the support. Example 5: Unimmobilized probe washing To remove the probes that were not immobilized on the support surface, the support was washed at room temperature using a 0.2% SDS (sodium dodecyl sulfate) solution and rinsed twice with distilled water. Next, the support was further washed with a sodium borohydride solution and then boiling distilled water. After washing at room temperature with a 0.2% SDS solution and then distilled water, water was removed from the surface of the support using a centrifuge, thereby resulting in a complete microarray.

Example 6: Hybridization

The biotin-labeled target DNA in Example 3 was heated to obtain single DNA strands and cooled at 4°C. A 10-μL hybridizing solution containing 1 -5μL of the target DNA solution was prepared. The hybridizing solution was portioned on the washed slide glass having the probes, covered with a cover slip such as to prevent air bubble formation therein, and reacted at 40°C for 30 minutes.

Example 7: Unhvbridized DNA washing In order to wash out the unhybridized DNA, the cover slip was removed using a solution mixture of 2X SSC (300mM NaCI, 30 mM Na-Citrate, pH 7.0) and 0.2% SDS, and the slide was washed with the solution mixture of 2X SSC/0.2% SDS, 2X SSC, and then 0.2X SSC. Water was removed from the washed slide using a centrifuge.

Example 8: Staining and assay

In order to check for the coupling of PCR products to the probes,

Cy5-streptavidin or Cy3-streptavidin (Amershiam Pharmacia Biotech,

USA) was diluted with 2X SSC and bovine serum albumin (BSA). About 40 μL of the dilution was portioned on the slide, covered with a cover slip, and reacted at 50°C for 20 minutes in the dark. The cover slip was removed using 2X SCC, and the slide was washed with 2X SSC and 0.2X SSC. Next, the coupling of PCR products to the probes on the slide was observed using a non-confocal laser scanner (GenePix 4000A, Axon Instruments, USA).

FIG. 2 shows the result of scanning the probes for simultaneously genotyping Mycobacteria species, differentiating M. tuberculosis strains, and detecting antibiotic-resistant strains, on the microarray after hybridization using one clinical sample through a single test. M. tuberculosis H37Rv strain hybridized to the probes on the microarray according to the present invention as a target DNA. As a result, among the probes for genotyping Mycobacteria species, specific hybridization occurred only in the

probes, MYO-02, in spots 1 and 21 of FIG. 1 , and the M. tuberculosis complex-specific probe, MTB-04. Among the probes for differentiating M. tuberculosis strains, the positive control probes in spots 49 and 93 of FIG. 1 , and the probes SPO-01 , SPO-03, SPO-04, SPO-05, SPO-06, SPO-08, SPO-11 , SPO-12, SPO-14, SPO-15, SPO-16, SPO-17, SPO-18, SPO-19, SPO-22, SPO-23, SPO-25, SPO-26, SPO-27, SPO-28, SPO-29, SPO-30, SPO-31 , SPO-32, SPO-37, SPO-38, SPO-39, SPO-40, SPO-41 , SPO-42, and SPO-43 in spots 50 through 92 of FIG. 1 specifically hybridized to the target DNA. As a result of using the standard M. tuberculosis H37Rv strain that is susceptible to all antibiotics in the antibiotic-resistance detection test, a hybridization reaction occurred in all positive control probes for detecting RMP-, IMH-, and PZA-resistance, respectively, in spots 22, 43, and 94 of FIG. 1. Among the RMP-resistance detecting probes, hybridization occurred in the probes rpo 511-WL, rpo 513-WQ, rpo 516-WD, rpo 522-WS, rpo 526-WH, rpo 531 -WD, and rpo 533-WL in spots 23, 25, 27, 29, 35, 38, respectively, of FIG. 1. INH-resistance was detected in the probes kat 315-WS and kat 463-WR in spots 44 and 47, respectively, of FIG. 1 , and PZA-resistance was detected in the probe pnc-11-W in spot 95 of FIG. 1.

FIGS. 3A and 3B show the scanned results of hybridization specific to the first probes for Mycobacteria species genotyping on the microarray according to the present invention when M. tuberculosis H37Rv strain and non-tuberculosis strain M. scrofυlaceum were used as the target DNA, respectively. In FIG. 3A, it is evident that hybridization occurred in the Mycobacterium genus-specific probes, MYC-02, in spots 1 and 21 of FIG. 1 and the M. tuberculosis-specific probe, MTB-04, in spot 2 of FIG. 1. As shown in FIG. 3B, hybridization occurred only in the Mycobacterium genus-specific probes, MYC-02, in spots 1 and 21 of FIG. 1 and the M.scrofulaceum-speάfic probe, SCO-01 , in spot 9 of FIG. 1.

FIG. 4A and 4B show the results of hybridization specific to the second probes for M. tuberculosis strain differentiation when M. tuberculosis H37Rv strain and M. bovis were used as the target DNA, respectively. Hybridization occurred in both positive control probes in spots 49 and 93 of FIG. 1. The M. tuberculosis H37Rv strain hybridized only to the probes SPO-01 , SPO-02, SPO-03, SPO-04, SPO-05, SPO-06, SPO-07, SPO-08, SPO-09, SPO-10, SPO-11 , SPO-12, SPO-14, SPO-15, SPO-16, SPO-17, SPO-18, SPO-19, SPO-22, SPO-23, SPO-24, SPO-25, SPO-26, SPO-27, SPO-28, SPO-29, SPO-30, SPO-31, SPO-37, SPO-38, SPO-39, SPO-40, SPO-41 , SPO-42, and SPO-43 in spots 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 63, 64, 65, 66, 67, 68, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 86, 87, 88, 89, 90, 91, and 92 of FIG. 1 , respectively, The M. bovis strain hybridized only to the probes SPO-01 , SPO-03, SPO-04, SPO-05, SPO-06, SPO-07, SPO-08, SPO-11 , SPO-12, SPO-12, SPO-13, SPO-17, SPO-18, SPO-19, SPO-20, SPO-21, SPO-22, SPO-23, SPO-24, SPO-25, SPO-26, SPO-27, SPO-28, SPO-29, SPO-30, SPO-31 , SPO-34, SPO-35, SPO-37, and SPO-38 in spots 50, 52, 53, 54, 55, 56, 57, 60, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 83, 84, 86, and 87 of FIG. 1 , respectively.

FIGS. 5A through 5F show the results of hybridization specific to the third probes for antibiotic-resistant Mycobacteria detection. As the results of a RMP-resistance test in FIGS. 5A and 5B when M. tuberculosis H37Rv strain and a RMP-resistant mutant with the substitution of tyrosine (TAC) for histidine (CAC) at codon 526 of the rpoB gene were used as the target DNA, respectively, hybridization occurred in the positive control probe which is a mixture of the individual probes for both strains. The RMP-susceptible M. tuberculosis H37Rv strain hybridized specifically to the wild-type probes for codons, i.e., rpo 511-WL, rpo 513-WQ, rpo 516-WD, rpo 522-WS, rpo 526-WH, rpo 531 -WD, and rpo 533-WL in spots 23, 25, 27, 30, 32, 38, and 41 of FIG. 1 , respectively. The RMP-resistant mutant with the substitution of tyrosine (TAC) for histidine (CAC) at codon 526 hybridized to rpo 511-WL, rpo 513-WQ, rpo 516-WD, rpo 522-WS, rpo 531 -WD, rpo 533-WL, and rpo 526-MY in spots 23, 25, 27, 30, 38, 41 , and 33 of FIG. 1 , respectively.

FIGS. 5C and 5D show the results of an INH-resistance test when M. tuberculosis H37Rv strain that is susceptible to INH and an INH-resistant mutant with the substitution of threonine (ACC) for serine (AGC) at codon 315 of the katG gene were used as the target DNA, respectively. As is apparent from FIGS. 5C and 5D, hybridization occurred in the positive control probe which is a mixture of the individual probes for both strains. The INH-susceptible M. tuberculosis hybridized only to the wild-type probes for codons 315 and 463, i.e., kat 315-WS and kat 463-WR in spots 44 and 47 of FIG. 1 , respectively. The INH-resistant mutant hybridized specifically to the probes kat 315-MT and kat 463-WR in spots 45 and 47 of FIG. 1 , respectively.

FIGS. 5E and 5F shows the result of a PZA-resistance test when M. tuberculosis H37Rv strain that is susceptible to PZA and an PZA-resistant mutant with the substitution of "G" for "A" in the pncA promoter-11 region were used as the target DNA, respectively. As is apparent from FIGS. 5E and 5F, the PZA-susceptible M. tuberculosis hybridized only to the positive control probe as a mixture of the individual PZA-resistance detection probes and the wild-type probe pnc-11-W in spot 95 of FIG. 1. The PZA-resistant mutant hybridized specifically to the positive control probe and the mutant detection probe pnc-11-M in spot 96 of FIG. 1.

Industrial Applicability As described above, the microarray according to the present invention includes Mycobacterium genotyping probes, M. tuberculosis strain differentiating probes, and antibiotic-resistance detecting probes together on a single support and thus can discriminate the Mycobacterium species and can detect 21 kinds of tuberculosis and non-tuberculosis Mycobacteria strains simultaneously through a single hybridization reaction. Since the plurality of probe sets are immobilized on the single support, a Mycobacterium genotyping test, a M. tuberculosis strain differentiation test, and an antibiotic-resistance detection test can be simultaneously performed on multiple clinical isolates, thereby reducing time consumption and costs. According to the present invention, clinical isolates as well as strain cultures can be directly applied so that the time for diagnosis is further reduced compared to conventional commercialized techniques. The Mycobacterium genotyping test, the M. tuberculosis strain differentiation test, and the antibiotic-resistance detection test can be performed within a short period of time, effectively and economically, using the microarray according to the present invention.

Claims

What is claimed is:

1. A microarray comprising: a support; a first probe for genotyping Mycobacteria species, immobilized on the support; a second probe for differentiating Mycobacterium tuberculosis strains, immobilized on the support; and a third probe for detecting antibiotic-resistant strains, immobilized on the support.

2. The microarray of claim 1 , wherein the first probe for genotyping Mycobacteria species, the second probe for differentiating Mycobacterium tuberculosis strains, and the third probe for detecting antibiotic-resistant strains are formed as one probe set, and a plurality of probe sets are immobilized on the support.

3. The microarray of claim 1 , wherein the first probe for genotyping Mycobacteria species includes a Mycobacterium genus-specific oligonucleotide based on the ITS (internal transcribed spacer) sequence, an oligonucleotide having the Mycobacterium tuberculosis species-specific ITS sequence, and an oligonucleotide having the Mycobacterium non-tuberculosis species-specific ITS sequence.

4. The microarray of claim 1 or 2, wherein the first probe for genotyping Mycobacteria species includes at least one of the oligonucleotides of SEQ ID NOs. 1 through 9, at least one of the oligonucleotides of SEQ ID NOs. 10 through 16, and at least one of the oligonucleotides of SEQ ID NOs. 17 through 89.

5. The microarray of claim 1 or 2, wherein the second probe for differentiating Mycobacterium tuberculosis strains includes an oligonucleotide of the direct repeat (DR) sequence specific to Mycobacterium tuberculosis strains.

6. The microarray of claim 1 or 2, wherein the second probe for differentiating Mycobacterium tuberculosis strains includes at least one of the oligonucleotides of SEQ ID NOs. 90 through 134.

7. The microarray of claim 1 or 2, wherein the third probe for detecting antibiotic-resistant strains is capable of simultaneously detecting resistance to rifampin, isoniazid, and pyrazinamide.

8. The microarray of claim 1 or 2, wherein the third probe for detecting antibiotic-resistant strains includes an oligonucleotide having a point mutation sequence of the rpoB gene that induces resistance to rifampin, an oligonucleotide having a point mutation sequence of the katG gene that induces resistance to isoniazid, and an oligonucleotide having a point mutation sequence of the pncA gene that induces resistance to pyrazinamide.

9. The microarray of claim 1 or 2, wherein the third probe for detecting antibiotic-resistant strains includes at least one of the oligonucleotides of SEQ ID NOs. 135 through 160, at least one of the oligonucleotides of SEQ ID NOs. 161 through 169, and at least one of the oligonucleotides of SEQ ID NOs. 170 through 173.

10. A method for simultaneously genotyping Mycobacteria species, differentiating Mycobacterium tuberculosis strains, and ■ ^' detecting antibiotic-resistant strains using the microarray of any one of claims 1 through 9.

11. A diagnostic kit for tuberculosis and non-tuberculosis Mycobacteria infections, comprising the microarray of any one of claims 1 through 9.

12. A primer or probe oligonucleotide for genotyping

Mycobacteria species, having at least one sense sequence or antisense sequence of SEQ ID NOs. 1 through 89.

13. A primer or probe oligonucleotide for differentiating Mycobacterium tuberculosis strains, having at least one sense sequence or antisense sequence of SEQ ID NOs. 90 through 134.

14. A primer or probe oligonucleotide for detecting antibiotic-resistant strains, having at least one sense sequence or antisense sequence of SEQ ID NOs. 135 through 173.