WO2010030049A1

WO2010030049A1 - Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time pcr using the same

Info

Publication number: WO2010030049A1
Application number: PCT/KR2008/005421
Authority: WO
Inventors: Jin Seok Kang; Young Suk Park; Jae Sung Lee
Original assignee: LG Life Sciences Ltd
Current assignee: LG Chem Ltd
Priority date: 2008-09-12
Filing date: 2008-09-12
Publication date: 2010-03-18
Anticipated expiration: 2011-03-12
Also published as: CN102149822A

Abstract

Provided is a composition for detection of M. tuberculosis complex or Mycobacterium genus, comprising (i) a primer and/or probe targeting IS6110 that is an M tuberculosis complex-specific gene, (ϋ) a primer and/or probe targeting rpoB that is a Mycobacterium genus-specific gene, and optionally (iϋ) a primer and/or probe targeting a plant-derived gene as an internal control. When multiplex real-time PCR is carried out using the composition of the present invention, it is possible to detect M tuberculosis complex as well as Mycobacterium genus by a single roundPCR assay. Therefore, the present invention enables rapid and easy clinical diagnosis with high reliability.

Description

COMPOSITION FOR DETECTION OF M. TUBERCULOSIS

COMPLEX OR MYCOBACTERIA GENUS AND SIMULTANEOUS

DETECTION METHOD FOR M. TUBERCULOSIS COMPLEX AND MYCOBACTERIA GENUS WITH MULTIPLEX REAL TIME PCR

USING THE SAME

FIELD OF THE INVENTION

The present invention relates to a composition for detection of Mycobacterium tuberculosis complex or Mycobacterium genus and a method for simultaneous detection of M tuberculosis complex and Mycobacterium genus by multiplex real-time PCR using the same. More specifically, the present invention relates to a composition for detection of M, tuberculosis complex or Mycobacterium genus, comprising (i) a primer and/or probe targeting IS6110 that is an M tuberculosis complex-specific gene, (ϋ) a primer and/or probe targeting rpoB that is a Mycobacterium genus-specific gene, and optionally (in) a primer and/or probe targeting a plant- derived gene as an internal control.

BACKGROUND OF THE INVENTION Tuberculosis is the world's leading infectious cause of death since before recorded history. The causative bacterial pathogen of tuberculosis is tuberculosis-causing bacteria which belong to the Mycobacterium genus and have a rod (bacillus) morphology, a thickness of 0.2-0.5 m and a length of 1-4 μsa.

The Mycobacterium genus encompasses a variety of pathogenic species causing respiratory diseases (including tuberculosis), leprosy (Hansen's disease), etc. in humans and animals. Up to date, more than 100 mycobacterial species have been identified (Shinnick TM et al, Mycobacterial Taxonomy. Eur J Clin Microbiol Infect Dis. 1994;13(11):884-901).

Mycobacterium tuberculosis is known as a major bacterial strain causing tuberculosis in humans. Rare cases of human tuberculosis are caused by Mycobacterium bovis. Mycobacterium leprae is known to cause leprosy, i.e. Hansen's disease (Shinnick TM and Good RC. Mycobacterial Taxonomy. Eur J Clin Microbiol Infect Dis. 1994;13(11):884-901).

Tuberculosis is one of the major causes of illness and death in economically underdeveloped countries. In Korea, it is estimated that 120,000 people are annually infected with tuberculosis pathogens. According to the statistical data of the Korea National Statistical Office (KNSO) in 2004, the incidence of new tuberculosis patients amounted up to 30,687 cases (64 cases per 100,000 people) in the survey of 2003, and Korea was shamefully ranked the highest among the 30 OECD Member countries in terms of tuberculosis mortality.

In recent years, the morbidity of atypical tuberculosis cases exhibiting clinical symptoms similar to those of tuberculosis infections is steadily growing due to infections of AIDS or other immunodeficient patients or immunocompromised infants with nontuberculous mycobacteria (NTM).

Examples of nontuberculous mycobacteria (NTM) may include M. avium-intracellulare complex or M. avium complex, M. fortuitum, M. chelonae, M. gordonae, M. szulagai, M kansasii, M φicanum, and M genavense (Barnes PF et al., Tuberculosis in patients with human immunodeficiency virus infectioa N Engl J Med. 1991;324(23): 1644-1650).

Many bacterial strains of the above-exemplified NTM exhibit multi-drug resistance against anti-tuberculosis drugs, which makes it difficult to obtain effective therapeutic effects on tuberculosis (Kam KM et al., Trends in Multidrug-Resistant Mycobacterium tuberculosis in Relation to Sputum Smear Positivity in Hong Kong, 1989-1999. Clin Infect Dis. 2002;34(3):324-

329).

In particular, Mycobacterium avium-intracellulare or M. avium complex (MAC), which is the most common species of NTM, is known to exhibit 10 to 100-fold lower susceptibility to first- line anti-tuberculosis drugs, when compared with tuberculosis bacteria For this reason, the American Thoracic Society (ATS) presents a guideline for diagnosis and treatment of NTM diseases (American Thoracic Society, Diagnosis and treatment of disease caused by nontuberculous mycobacteria Am J Respir Crit Care Med. 1997; 156(2): S 1-S25).

That is, pathogenic symptoms of NTM diseases are very similar to those of tuberculosis infections, but therapeutic drugs for treatment of both diseases may be completely different therebetween. In order to provide reasonable therapeutic regimens corresponding to individual patients infected with tuberculosis or non-tuberculosis mycobacteria, there is an essential need for early and accurate detection and diagnosis of tuberculosis and nontuberculosis mycobacteria of concerned patients.

Conventional methods for diagnosis of tuberculosis may include examination of patients' clinical symptoms, tuberculin skin test, X-ray photography and bacteriological test. The tuberculin skin test is the simplest means for identifying patients infected with tuberculosis, but disadvantageously may often produce false-negative results under anergy-inducing conditions due to severe tuberculosis, rubeola (measles) and immunosuppression. Further, the efficacy of X-ray diagnosis is determined by the reader's ability and an average of 25% of patients diagnosed positive by X-ray examination are diagnosed negative by other diagnostic tests. Thus, the diagnosis of tuberculosis by X-ray photography depends on the finding of abnormal shadows and accurate interpretation of the abnormal shadows by the reader.

The bacteriological test, Ie. the detection of tuberculosis bacteria, is a reliable method for diagnosis of tuberculosis infection and may include a smear test, a culture test, a molecular diagnostic test, etc.

The smear test generally employs Ziehl-Neelsen staining which is a special technique used to identify acid-fast organisms, mainly mycobacteria. Although it is a simple and rapid diagnostic technique, identification of tuberculosis and nontuberculous mycobacteria is difficult and detection sensitivity is also low.

The culture test is advantageous to provide high detection sensitivity for accurate diagnosis and identification of tuberculosis bacteria That is, it is possible to detect pathogenic bacteria even at a very low concentration of about 10 bacteria/mL of a test sample. Unfortunately, this method is not therapeutically favorable for the treatment of tuberculosis patients, due to requirements for long-term culture of about 4 to 8 weeks and examination by well-trained specialists to get the test results.

A BACTEC method (Becton Dickinson, USA) is a novel method for diagnosis of tuberculosis that radioisotopicaUy measures an amount of ¹⁴CO₂ produced by the metabolism of bacteria inoculated onto a liquid medium containing C¹⁴-palmitate and expresses the measured amount as a growth quotient. This method takes an average of 16 days to get the results. However, there is a problem that facilities and skilled persons for safe handling and management of radioisotopes are required.

An electrophoresis-based polymerase chain reaction (PCR) is a method capable of rapidly and accurately diagnosing tuberculosis by amplification of specific gene regions within a short period of 2 to 3 hours. The PCR assay is a useful technique enabling the detection of tuberculosis bacteria in clinical samples with high sensitivity and specificity of 95% or more within one day (Wilson SM et al., Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis. J Clin Microbiol. 1993; 31(4):776-78).

However, the PCR assay has shortcomings such as high risk of carry-over contamination and need for PCR technicians (Noordhoek GT et al., Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol. 1994;32(2):277-284).

With regard to the diagnosis of tuberculosis, Ihe PCR assay exhibits high sensitivity and specificity particularly in the smear-positive sample. Therefore, the possibility should be considered that the sample of interest is likely to contain NTM when it is acid-fast smear-positive and PCR- negative.

In the United States where the incidence of NTM pulmonary diseases is high, it is recommended to make a diagnosis as follows. When the acid-fast smear-positive sample is PCR- positive, a subject of interest may be tentatively diagnosed with pulmonary tuberculosis. When the acid-fast smear-positive sample is PCR-negative, a subject of interest may be tentatively diagnosed with infection of NTM when the sample is again found to be PCR-negative even after the presence/absence of a PCR inhibitory material in the sample is confirmed. The final diagnosis of

NTM infection takes a culture period of 4 to 8 weeks (Centers for Disease Control and Prevention (CDC). Update: Nucleic acid amplification tests for tuberculosis. MMWR Morb Mortal WkIy Rep

2000; 49:593-4). Even though exposure of a subject to NTM species may be estimated according to such a guideline, it will be more clinically useful if NTM can be directly identified.

In Korea, the morbidity of tuberculosis is high whereas the incidence of NTM disease is low. Therefore, when a test sample of the subject is acid-fast smear-positive, most of suspected cases are generally regarded as having tuberculosis, followed by appropriate medication of antituberculosis drugs. However, an isolation rate of NTM and the incidence of NTM diseases have recently been increasing in Korea Immune-compromised individuals may be susceptible to NTM diseases, whereas the diagnosis of NTM infection is not easy. Further, it is difficult to effectively treat NTM diseases, due to high drug resistance of NTM species. In addition, NTM diseases also exhibit a high risk of recurrence (Scientific Committee in Korean Academy of Tuberculosis and Respiratory Diseases. National survey of mycobacterial diseases other than tuberculosis in Korea. Tuberc Respir Dis 1995;42:277-94.). For these reasons, there is an urgent need for development of a diagnostic method which is capable of detecting and identifying NTM species. As a currently available method of detecting NTM species, mention may be made of an

AFB staining method. As molecular biological techniques for identification of mycobacterial species, there are currently commonly used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using restriction enzymes and PCR-hybridization using specific probes.

The above-mentioned methods exhibit low detection sensitivity and therefore disadvantageously require previous cell culture prior to practical testing or should involve complicated experimental steps. Consequently, these conventional methods are not favorable for early detection of M. tuberculosis complex and Mycobacterium genus.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a primer and/or probe having excellent sensitivity for an M. tuberculosis complex-specific gene IS6110, which is intended for accurate diagnosis of M tuberculosis.

It is another object of the present invention to provide a primer and/or probe having excellent sensitivity by the detection of a high-specificity gene region from an rpoB gene common to all species of the Mycobacterium genus, such that the presence of Mycobacterium genus can be detected. It is yet another object of the present invention to provide a method which is capable of achieving simultaneous detection of M tuberculosis complex and nontuberculous mycobacteria (NTM) by multiplex real-time PCR using the aforesaid primer and/or probe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table depicting partial base sequences of rpoB genes of mycobacterial species conventionally known in the art;

FIG. 2 is a photograph showing the multiplex real-time PCR results of M tuberculosis complex-positive samples using an analysis composition in accordance with the present invention;

FIG. 3 is a photograph showing the multiplex real-time PCR results of Mycobacterium genus-positive samples using an analysis composition in accordance with the present invention; and

FIG. 4 is a photograph showing the multiplex real-time PCR results of negative samples using an analysis composition in accordance with the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

L Primers for detection of M. tuberculosis complex or Mycobacterium genus In one aspect, the present invention provides a composition for detection of M. tuberculosis complex or non-tuberculosis mycobacteria, comprising at least one primer selected from the group consisting of: a primer containing a base sequence as set forth in SEQ ID NO: 1 ; a primer containing a base sequence as set forth in SEQ ID NO: 2; a primer containing a base sequence as set forth in SEQ ID NO: 3 ; and a primer containing a base sequence as set forth in SEQ ID NO: 4.

As used herein, the term "a primer containing a base sequence as set forth in SEQ ID NO: x" is intended to encompass a base sequence with a sequence homology of 95% or more. The reason is as follows. If two or more bases are altered when a primer is 20bp in length, lowering of a melting temperature of the primer is 5 ^°C or more, which consequently leads to undesirable results. On the other hand, if a single base is changed, it is possible to obtain substantially the same results by slightly lowering an annealing temperature in a PCR process.

For brevity and convenience of explanation in the context of the present invention, "a primer containing the base sequence of SEQ ID NO: x" is simply designated as "a primer of SEQ ID NO: x.

In the context of the present invention, the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 are primers that are intended to target an IS6110 gene region which is an M. tuberculosis complex-specific gene region.

Generally even though "tuberculosis bacteria" means human-type tuberculosis bacilli, Ie. Mycobacterium tuberculosis in a narrow sense, this term in the context of the present invention is intended to encompass Mycobacterium bovis, Mycobacterium microti and Mycobacterium qfricanum as well as M. tuberculosis in a broad sense and is used interchangeably with the term "M tuberculosis complex" or "TB complex", if necessary.

The IS6110 gene is an insertion sequence which is found in M. tuberculosis complex including human-type Mycobacterium tuberculosis and bovine-type Mycobacterium bovis. The M. tuberculosis complex is known to contain 10 to 12 copies of IS6110 which are commonly used in the PCR diagnosis of tuberculosis (Thierry, D., et al., Clin Microbiol. 1990;28(12):2668-2673). However, KENT et al. reported that IS6110 shows a difference in the false-positive risk, depending on a selection position of the primer (J. Clin Microbiol 1995;33(9):2290-2293).

To this end, inventors of the present invention conducted a study on a low false-positive risk regions using base sequence analysis and found, as will be confirmed in the following

Examples, that the primer of SEQ DD NO: 1 and the primer of SEQ ID NO: 2 are primer base sequences capable of amplifying only an IS6110 gene of M tuberculosis complex and exhibit very high sensitivity of more than 97% and a positive predictive value of more than 99% for the IS6110 gene region of the M tuberculosis complex, thus confirming extremely low risk of false positives. To the best of our knowledge, there is yet no report of such primers with high sensitivity and positive predictive value.

Therefore, when either or both of the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 are used in the diagnosis of tuberculosis infection, it is advantageously possible to detect the M. tuberculosis complex with very high reliability. Meanwhile, Doucet-Populaire and colleagues reported that about 7% of nontuberculous mycobacteria (NTM) contain fragments or analogues of IS6110, thus potentially presenting the risk of false positives (Tuber Lung Dis 1996; 77(4): 358-62).

Therefore, identification of the Mycobacterium genus is important to confirm the presence/absence of nontuberculous mycobacteria In this connection, the rpoB gene is in common among the Mycobacterium genus and it is therefore known that the presence of rpoB can be used for identification of mycobacterial species (Lee, Hye- Young, et al., Korean Patent Application

PublicationNo. 10-2001-0038701).

However, the above Korean Patent employs a PCR-RFLP that disadvantageously requires prior cell culture due to low sensitivity or involves complicated experimental steps. As a consequence, this method is not suitable for prompt detection of M tuberculosis complex and Mycobacterium genus at the early stage.

To this end, the inventors of the present invention intend to employ an analysis method that is capable of rapidly and easily performing real-time PCR with high sensitivity for the Mycobacterium genus.

However, available primers known hitherto for detection of the rpoB gene form a very large amplification product (340-360bp), so they are not suitable for real-time PCR.

To cope with this problem, the present inventors developed primers forming an amplification product having a size suited for the real-time PCR. That is, the primer of SEQ ID NO: 3 and the primer of SEQ ID NO: 4 are primers for targeting of the Mycobacterium genus-specific gene rpoB and form PCR amplification products having a length of lOObp or less. Advantageously, use of these primers results in a highly reliable, rapid, and accurate detection of Mycobacterium genus.

Optionally, the composition for detection of M tuberculosis complex or Mycobacterium genus in accordance with the present invention may further comprise an internal control primer. Incorporation of the internal control is intended to confirm problems of false negatives, Ie. whether PCR was correctly carried out. For this purpose, a suitable gene may be optionally selected which is normally expressed irrespective of the presence/absence of M. tuberculosis or other mycobacterial species in the sample of interest.

In one embodiment of the present invention, the aforesaid internal control primer may be a plant-derived gene-specific primer that is added to all the test samples.

The plant-derived gene may be preferably a lectin gene. The lectin gene-specific primer may be a primer containing a base sequence as set forth in SEQ ID NO: 28 or a primer containing a base sequence as set forth in SEQ ID NO: 29.

Table 1 below shows base sequences for the primers of SEQ DD NOs: 1 to 4, 28 and 29.

[Table 1]

Base sequences of primers

As reviewed before, the present invention provides a composition for detection of M. tuberculosis complex comprising a sense primer containing a base sequence as set forth in SEQ DD NO: 1 and/or an antisense primer containing a base sequence as set forth in SEQ ID NO: 2, as novel M. tuberculosis complex IS6110-specific primers.

Further, the present invention provides a composition for detection of Mycobacterium genus comprising a sense primer containing a base sequence as set forth in SEQ DD NO: 3 and/or an antisense primer containing a base sequence as set forth in SEQ DD NO: 4, as novel mycobacterial rpoB-specific primers.

The mycobacterial rpoB-specific primers form PCR amplification products having a length of less than 1 OObp suitable for real-time PCR.

In one embodiment of the present invention, the mycobacterial species may be M. tuberculosis or M bovis. The detection reliability of tuberculosis bacteria can be further improved by co-analysis of the rpoB gene in combination with the IS6110 gene of the M. tuberculosis complex.

In another embodiment of the present invention, the mycobacterial strain may be nontuberculous mycobacteria (NTM). In this case, it is advantageously possible to confirm the presence/absence and kinds of nontuberculous mycobacteria other than the M. tuberculosis complex, through base sequencing of the rpoB gene.

Among the nontuberculous mycobacteria, those having a known rpoB gene sequence are shown in FIG. 1.

In addition to the mycobacterial species given in FIG. 1, the present inventors partially analyzed base sequences of rpoB genes of the following mycobacterial strains: M. acapulsewis, M. flavescens, M. gastin, M intracellulare, M. kansasii, M. pulveris, M. simiae, M. xenopi Schwabacher, M. austroafricamm, M. celatum, M. gastri, M. gordonae, M. agri, Masiaticum, M. cekatum, M. diernhoferi, M.fortuitum, M. nonchromogenicum, Mphlei, and M. genavense.

Further, the present inventors confirmed that the primers having base sequences of SEQ

ID NOs: 3 and 4 are effective for identification of the following 42 mycobacterial species and exhibit specificity for them.

Probes for detection of M. tuberculosis complex or Mycobacterium genus

In another aspect, the present invention provides a composition for detection of M. tuberculosis complex or Mycobacterium genus, comprising at least one probe selected from the group consisting of: a probe containing a base sequence as set forth in SEQ ID NO: 5; a probe containing a base sequence as set forth in SEQ ID NO: 6; and a probe containing a base sequence as set forth in SEQ ID NO: 7.

Optionally, the composition may further comprise an internal control probe. The internal control probe is preferably a probe specific for a lectin gene that is a plant-derived gene. The internal control probe may be a probe containing a base sequence as set forth in SEQ ID NO: 30. As used herein, the term "a probe containing a base sequence as set forth in SEQ ID NO: x" is intended to encompass a base sequence with a sequence homology of 95% or more. For brevity and convenience of explanation in the context of Ihe present invention, "a probe containing the base sequence of SEQ ID NO: x" is simply designated as "a probe of SEQ ID NO: x.

The probe of SEQ ID NO: 5 specifically reacts with the IS6110 gene region, and the probes of SEQ ID NOs: 6 and 7 are probes that react in common with the rpoB gene of any member of the Mycobacterium genus that can be detected.

In this manner, it is possible to further clearly detect whether the mycobacterial strains in a sample of interest are tuberculosis-causing bacteria or not, using IS6110- and rpoB-specific probes. By ∞nfirming the presence/absence of the rpoB gene, it is also possible to confirm the presence/absence of nontuberculous mycobacteria

In particular, the IS6110- or rpoB-specific probes in accordance with the present invention can be usefully used in PCR quantitative analysis by TaqMan assay or molecular beacon assay.

In one preferred embodiment of the present invention, the aforesaid probe is labeled with a fluorescent dye at 5' and 3' ends thereof. In this connection, the 5-labeled fluorescent dye (reporter) and the 3-labeled fluorescent dye (quencher) may be ones exhibiting mutual interference therebetween. Accordingly, when the probe is combined with the IS6110 or rpoB gene in the sample, color development of the probe is limited. Then, when the probe is decomposed with PCR, the 3-labeled fluorescent dye is quenched whereas the 5-fluorescent dye undergoes color development. There is no particular limit to the 5'-labeled fluorescent dye. Examples of the 5'-labeled fluorescent dye may include, but are not limited to, 6-carboxyfluorescein (FAM), hexachloro-6- carboxyfluorescein (HEX), tetrachloro-ό-carboxyfluorescein, and Cyanine-5 (Cy5).

In addition, examples of the 3'-labeled fluorescent dye may include, but are not limited to, 6κ^box^e1rarnethyl-rhodamine (TAMRA) and black hole quencher- 1,2,3 (BHQ-1,2,3).

Table 2 below shows base sequences for the probes of SEQ ID NOs: 5 to 7, and 30.

[Table 2]

Base sequences of probes

Composition and Kit for real-time PCR assay The present invention further provides a composition for analysis of M. tuberculosis complex and/or Mycobacterium genus by real-time PCR, comprising:

at least one primer selected from the group consisting of: a primer containing a base sequence as set forth in SEQ ID NO: 1 ; a primer containing a base sequence as set forth in SEQ ID NO: 2; a primer containing a base sequence as set forth in SEQ ID NO: 3 ; and a primer containing a base sequence as set forth in SEQ ID NO: 4; and

at least one probe selected from the group consisting of: a probe containing a base sequence as set forth in SEQ ID NO: 5; a probe containing a base sequence as set forth in SEQ ID NO: 6; and a probe containing a base sequence as set forth in SEQ ID NO: 7.

The composition may further comprise an internal control primer and an internal control probe to prevent false negatives.

The internal control primer may be a sense primer containing a base sequence as set forth in SEQ ID NO: 28 or an antisense primer containing a base sequence as set forth in SEQ ID NO: 29. The internal control probe may be a probe containing a base sequence as set forth in SEQ ID NO: 30.

That is, the quantitative analysis composition in accordance with the present invention comprises the primer(s) for detection of tuberculosis bacteria or nontuberculous mycobacteria, the probφ) for detection of tuberculosis bacteria or nontuberculous mycobacteria, and optionally, the internal control primer and probe. Using the composition in accordance with the present invention, the real-time PCR assay can be carried out involving simultaneous amplification of two or three genes in a single tube in conjunction with DNA molecules extracted from a clinical sample, and real-time analysis and detection of the resulting amplification products.

In one embodiment of the present invention, the composition may comprise:

(i) an M. tuberculosis complex IS6110-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 1 and the antisense primer containing a base sequence of SEQ ID NO: 2, and the probe containing a base sequence of SEQ ID NO: 5;

(ϋ) a Mycobacterium genus rpoB-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 3 and the antisense primer containing a base sequence of SEQ ID NO: 4; and the probe containing a base sequence of SEQ ID NO: 6 and/or the probe containing a base sequence of SEQ ID NO: 7;

(iϋ) an internal control-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 28 and the antisense primer containing a base sequence of SEQ ID NO: 29; and the probe containing a base sequence of SEQ ID NO: 30; and

(iv) a PCR reaction mixture containing buffer, DNA polymerase, dNTP and sterile distilled water.

As described above, the analysis composition of the present invention is comprised of three pairs of primers and three probes. Using the aforesaid composition, the present inventors carried out multiplex real-time PCR As a result, it was confirmed that tuberculosis bacteria and other mycobacterial strains in the analyte sample can be detected and diagnosed with substantially complete exclusion of the risk of false positivity.

Specifically, the mixture (i) is capable of amplifying only the M. tuberculosis complex and includes a primer having high sensitivity of more than 97% and a positive predictive value of more than 99% for the IS6110 gene region and a probe that specifically binds to the IS6110 gene region which was amplified using the aforesaid primer.

Further, the mixture (ii) is capable of amplifying the rpoB gene that is a Mycobacterium genus-specific gene and includes a primer forming an rpoB gene region having a length suitable for real-time PCR-based assay and a probe that specifically binds to the rpoB gene region which was amplified using the aforesaid primer.

The aforesaid probe binds to the rpoB gene of any member of the Mycobacterium genus that is detectable, whereby it is possible to detect the presence/absence of mycobacterial species through binding of the probe to the rpoB gene.

If desired, the composition may further comprise one or more probes containing an rpoB gene region having a different base sequence extracted from different mycobacterial species. In this case, mycobacterial species corresponding to the additionally incorporated probe can be identified.

Hence, when an assay kit comprising the aforesaid composition is used, it is possible to more conveniently and precisely quantify tuberculosis-specific genes and confirm the presence/absence of nontuberculous mycobacteria The composition of the present invention can be widely used for accurate diagnosis of tuberculosis infection. The real-time PCR assay may be carried out using commercially available real-time PCR equipment. Examples of the real-time PCR equipment may include, but are not limited to, SLAN real-time PCR detection system (LG Life Sciences, Korea), LightCycler™ (Roche, Germany), ABI

PRISM™ 7000/7700 (Applied Biosystems, USA), iCycler™ (Bio-Rad, USA), Rotor-Gene^"™ (Corbett, Australia), and Opticon™ (PharmaTech, USA).

4 Analysis of M. tuberculosis complex or Mycobacterium genus

The analysis composition in accordance with the present invention may be used in qualitative analysis to confirm the presence/absence of M tuberculosis complex or Mycobacterium genus, or otherwise may be used in quantitative analysis of M tuberculosis complex or Mycobacterium genus. That is, the present invention provides a method for quantitative or qualitative analysis of tuberculosis bacteria or nontuberculous mycobacteria, using the aforesaid analysis composition.

The qualitative analysis intended for identification of the presence/absence of M. tuberculosis complex or Mycobacterium genus can be carried out by confirming a time point at which a peak is observed by means of real-time PCR using the analysis composition.

Further, the quantitative analysis can be carried out by comparison with a standard curve. In one preferred embodiment of the present invention, the quantitative analysis can be carried out according to the following procedure steps using the analysis composition: (1) mixing the analysis composition with bacterial DNA extracted from a sample of interest to prepare a gene analysis sample;

(2) subjecting the gene analysis sample of Step (1) to real-time PCR to obtain a PCR product;

(3) quantifying the PCR product of Step (2) to obtain a quantitative curve; and

(4) quantifying IS6110-, rpoB- and internal control-specific genes in the bacterial DNA of the sample, using the quantitative curve.

That is, the sample-extracted bacterial DNA is added to the quantitative analysis composition comprising three primer pairs and three probes including an M. tuberculosis complex IS6110-specific primer pair and probe, a mycobacterium genus rpoB-specific primer pair and probe, and an internal control-specific primer pair and probe, and real time PCR is then carried out. As a result, each of IS6110-, rpoB- and internal control (lectin gene)-specific primer pairs and probes is attached to DNA molecules to result in amplification of a PCR product.

When TaqMan assay is used as a method for quantification of the PCR product, the IS6110, rpoB and internal control-specific genes can be quantified by rønfirming amplification of the genes through the fluorescence emitted upon decomposition of the probes attached to the IS6110, rpoB and lectin genes.

Mycobacterium genus rpoB-specific nucleic acids Further, the present invention provides an rpoB-specific nucleic acid of the Mycobacterium genus, the nucleic acid comprising at least one selected from the group consisting of base sequences of the following SEQ ID NOs: 8 to 27:

M acapulsensis-speάSc nucleic acid containing a base sequence as set forth in SEQ ID NO: 8;

Mflavescens-spectfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 9;

M gastin-sped&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 10;

M intracellulare-speci&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 11;

M. kansasii-sp&ά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 12;

M. pulveris-specϊ&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 13; M. simiae-speάΑc nucleic acid containing a base sequence as set forth in SEQ ID NO: 14;

M. xenopi Schwabacher-specific nucleic acid containing a base sequence as set forth in SEQIDNO: 15; M austroqfricamm-sped&c nucleic acid containing a base sequence as set forth in SEQ IDNO: 16;

M celatum-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 17; M. gastri-sp&ά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 18;

M. gordonae-speάfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 19;

M. αgπ-specific nucleic acid containing a base sequence as set forth in SEQ ID NO: 20;

M asiaticum-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 21;

M cekatum-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO:

22;

M. diernhoβri-specϊfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 23; M. fortuitum-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO:

24;

M nonchromogenicum-speάfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 25; M phlei-sp&ά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 26; and

M genavense-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 27.

All of the above-exemplified nucleic acids contain the rpoB gene. Using these base sequences, it is possible to construct primers or probes that react in common with the Mycobacterium genus or it is possible to easily construct primers or probes having specificity corresponding to each mycobacterial species.

EXAMPLES

Now, the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1: Construction of primers and probes for multiplex real-time PCR

It was confirmed that IS6110-specific primers and probes used herein were primer and probe sequences capable of amplifying only the M tuberculosis complex by analyzing the DNA sequence, deposited under accession number NC000962 in GenBank (www.ncbi.nlm.nih.gov') managed by the National Center for Biotechnology Information (NCBI), National Institutes of Health (NIH), using DNAsis which is a gene analysis program available from Hitachi Software, picking the appropriate sequence, and then analyzing the DNA sequence again with BLAST (www.ncbi.nlm.nih.gov/BLASTΛ.

Further, it was confirmed that rpoB-specific primers and probes used herein were primer and probe sequences capable of amplifying only the Mycobacterium genus, by obtaining a

Mycobacterium rpoB gene from the GenBank/Entrez Protein database, analyzing a conserved region of the rpoB gene using the software Clustal-X (see FIG. 1), picking the appropriate sequence, and then analyzing the DNA sequence again with BLAST (www.ncbi.nlm.nih.gov/BLAST/).

Thereafter, unknown other base sequences were obtained using standard strains (see FIG. 2). It was confirmed that the sequenced primers are also capable of amplifying genes of the standard strains which were newly obtained.

Base sequences of the plant-derived gene-specific primer and probe were obtained from patent references published in the art.

Example 2; Synthesis of primers

The primers analyzed in Example 1 were synthesized on request by Metabion (Germany) according to the method such as "Synthesis of Oligonucleotide" described in a paragraph 10.42 of Molecular Cloning 3^rd ed (Sambrook and Russell, Cold Spring Harbor Laboratory Press, New York, USA, 2001).

Example 3: Extraction of mycobacterial DNA from clinical sample or standard strain culture medium

1 to 2 mL of sputum of a suspected tuberculosis patient or standard strain culture medium and an equal amount of 4N NaOH were placed in a 15 mL tube, sufficiently stirred, and then centrifuged at 4,000 rpm for 20 minutes. The supernatant was discarded, and 10 mL of a PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) was added to the precipitate, and the resulting mixture was thoroughly stirred and centrifuged at 4,000 rpm for 20 minutes. The supernatant was again discarded. The precipitate was transferred to a 1.5 mL tube and 1 mL of a PBS buffer was added thereto. The resulting mixture was stirred and centrifuged at 13,000 rpm for 5 minutes. The supernatant was again discarded. 50 to 100 μi of 5% (w/v) Chelex 100 resin (Bio- Rad) was added to the precipitate, and the resulting mixture was heated at 100^°C for 20 minutes and centrifuged at 13,000 rpm for 3 minutes. Supernatant with extracted DNA was used as template for PCR amplification.

Example 4: Multiplex real-time PCR assay using primers and probes (1) PCR compositions were prepared according to a composition formula given in Table 3 below, and PCR was carried out in a SLAN real-time PCR detection system (LG Life Sciences, Korea) under the reaction conditions set forth in Table 4 below.

(2) The reaction products were determined on a real-time basis. After the reaction was complete, the results were analyzed using a SLAN 7.0 program.

(3) It was determined as follows: FIG.2: M. tuberculosis complex-positive, FIG. 3: Mycobacterium genus-positive, and FIG.4: Negative.

[Table 3]

Real-time PCR compositions

[Table 4]

Real-time PCR conditions

Example 6; Diagnostic comparison of conventional culture method (MGIT, BD, USA) and Inventive kit for M. tuberculosis complex and Mycobacterium genus

Diagnosis of M. tuberculosis complex and Mycobacterium genus was compared between a conventional culture method (MGIT, BD, USA) and a kit of the present invention. The comparative culture method was carried out according to the manufacturer's instructions. For the cultured positive sample, the mycobacterial species was confirmed with an amplicor MTB kit (Roche Diagnostic Systems, NJ, USA) and base sequence analysis. The results obtained are given in Table 5 below.

[Table 5]

Comparison experimental results

A. Result table

* TB: M. tuberculosis complex/NTM: nontuberculous mycobacteria

B. Sensitivity and Specificity

As used herein, the term "sensitivity" refers to the proportion of people with disease in whom the test result is positive. The term "specificity" refers to the proportion of people without disease who have a negative test result. The term "positive predictive value" as used herein refers to the probability of the disease being present, among those with positive diagnostic test results. The term "negative predictive value" as used herein refers to the probability that the disease was absent, among those whose diagnostic test results were negative.

According to the results of Table 5, when RT-PCR was carried out using the composition in accordance with the present invention, it was clearly confirmed that the substantially comparable reliability was obtained upon comparison with the culture method which is currently the highest reliability assay of tuberculosis bacteria and nontuberculous mycobacteria In particular, the sensitivity to M. tuberculosis complex was 97% or higher, and the specificity was 99%, thus representing that the composition of the present invention enables a substantially accurate identification of tuberculosis bacterial species. Example 7: Diagnostic comparison of conventional AFB staining and Inventive kit for M. tuberculosis complex and Mycobacterium genus

The samples obtained in Example 3 were respectively subjected to AFB staining which has been conventionally used in the confirmation of nontuberculous mycobacteria (NTM) and RT- PCR using a kit of the present invention. For comparison, the results obtained are given in Table 6 below.

[Table 6]

In Table 6, (-) represents NTM-negative, (+) represents NTM-positive, and an increasing number of (+) represents higher concentration of NTM.

As can be seen from Table 6, all the samples which were determined NTM-positive by

AFB staining were also NTM-positive in RT-PCR results obtained according to the present invention. On the other hand, it was confirmed that, among the samples which were determined to be NTM-negative by AFB staining, no fewer than three samples were found NTM-positive according to the RT-PCR results of the present invention. From these results, when the composition of the present invention is used, it is possible to identify NTM with significantly higher sensitivity and reliability, when compared with a conventional AFB staining method which is an NTM assay method widely used in the art.

Example 8: Inventive RT-PCR assay for various mycobacterial species

Analogously to Examples 3 and 4, RT-PCR was carried out for a total of 42 mycobacterial species. The results obtained are given in Table 7 below.

[Table 7]

In Table 7, a value of less than 35 represents "Mycobacterium-iposiiάyQ" and IC represents "internal control". In addition, Q represents a threshold cycle number, Ie. the number of cycles that pass through a critical value when RT-PCR was performed, and therefore No Q represents that there is no threshold cycle.

As can be seen from Table 7, the results of RT-PCR in accordance with the present invention revealed that it was tuberculosis-positive for M. tuberculosis H37Rv, M. bovis, M. bovis BCG, M. africanum and M microti, all of which belong to the M tuberculosis complex, whereas it was λfycobacterium-posiύve for the remaining Mycobacterium species. Therefore, it was clearly confirmed that RT-PCR using the analysis composition of the present invention is useful for the detection of M tuberculosis complex and Mycobacterium genus.

INDUSTRIAL APPLICABILITY

As apparent from the above description, when real-time PCR is carried out using an analysis composition comprising specific primers and probes in accordance with the present invention, it is possible to perform detection of Mycobacterium tuberculosis complex and Mycobacterium genus with excellent sensitivity and specificity within a shorter period of time, as compared to a conventional method.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

WHAT IS CLAIMED IS

1. A composition for detection of M. tuberculosis complex or Mycobacterium genus, comprising at least one primer selected from the group consisting of: a primer containing a base sequence as set forth in SEQ ID NO: 1 ; a primer containing a base sequence as set forth in SEQ ID NO: 2; a primer containing a base sequence as set forth in SEQ ID NO: 3; and a primer containing a base sequence as set forth in SEQ D NO: 4.

2. The composition according to claim 1 , further comprising an internal control primer.

3. The composition according to claim 2, wherein the internal control primer is a plant- derived gene-specific primer.

4. The composition according to claim 3, wherein the plant-derived gene is a lectin gene and the plant-derived gene-specific primer is a primer containing a base sequence as set forth in SEQ ID NO: 28 or a primer containing a base sequence as set forth in SEQ ID NO: 29.

5. A composition for detection of M. tuberculosis complex comprising a sense primer containing a base sequence as set forth in SEQ ID NO: 1 and/or an antisense primer containing a base sequence as set forth in SEQ ID NO: 2, as primers specific for an IS6110 gene of M. tuberculosis complex.

6. A composition for detection of Mycobacterium genus comprising a sense primer containing a base sequence as set forth in SEQ ID NO: 3 and/or an antisense primer containing a base sequence as set forth in SEQ ID NO: 4, as primers specific for an rpoB gene of Mycobacterium genus.

7. The composition according to claim 6, wherein the primers specific for the rpoB gene of the Mycobacterium genus form PCR amplification products having a length of less than 1 OObp.

8. A composition for detection of M. tuberculosis complex or Mycobacterium genus, comprising at least one probe selected from the group consisting of: a probe containing a base sequence as set forth in SEQ ID NO: 5; a probe containing a base sequence as set forth in SEQ ID NO: 6; and a probe containing a base sequence as set forth in SEQ ID NO: 7.

9. The composition according to claim 8, wherein the probe is labeled with a fluorescent dye at 5' and 3' ends thereof, and the 5'-labeled fluorescent dye (reporter) and the 3-labeled fluorescent dye (quencher) exhibit mutual interference therebetween.

10. The composition according to claim 9, wherein the 5'-labeled fluorescent dye is selected from the group consisting of 6-carboxyfluorescein (FAM), hexachloro-ό-carboxyfluorescein (HEX), tetrachloro-ό-carboxyfluorescein, and Cyanine-5 (Cy5), and the 3'-labeled fluorescent dye is 6-(^boxytetramethyl-rhodamine (TAMRA) orblackhole quencher-1,2,3 (BHQ-1,2,3).

11. A composition for analysis of M. tuberculosis complex or Mycobacterium genus by realtime PCR, comprising:

at least one primer selected from the group consisting of: a primer containing a base sequence as set forth in SEQ ID NO: 1 ; a primer containing a base sequence as set forth in SEQ ID NO: 2; a primer containing a base sequence as set forth in SEQ ID NO: 3; and a primer containing a base sequence as set forth in SEQ ID NO: 4; and

at least one probe selected from the group consisting of: a probe containing a base sequence as set forth in SEQ ID NO: 5; a probe containing a base sequence as set forth in SEQ ED NO: 6; and a probe containing a base sequence as set forth in SEQ ID NO: 7.

12. The composition according to claim 11 , further comprising an internal control primer and an internal control probe.

13. The composition according to claim 12, wherein the internal control primer is a sense primer containing a base sequence as set forth in SEQ ID NO: 28 or an antisense primer containing a base sequence as set forth in SEQ ID NO: 29, and the internal control probe is a probe containing a base sequence as set forth in SEQ E) NO: 30.

14. The composition according to claim 11 , wherein the composition comprises:

(i) an M tuberculosis complex IS6110-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 1 and the antisense primer containing a base sequence of SEQ ID NO: 2, and the probe containing a base sequence of SEQ ID NO: 5;

(ϋ) a Mycobacterium genus rpoB-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 3 and the antisense primer containing a base sequence of SEQ E) NO: 4; and the probe containing a base sequence of SEQ E) NO: 6 and/or the probe containing abase sequence of SEQ IDNO: 7;

(iii) an internal control-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 28 and the antisense primer containing a base sequence of SEQ ID NO: 29; and the probe containing a base sequence of SEQ E) NO: 30; and

15. A kit for analysis of M tuberculosis complex or Mycobacterium genus comprising the composition of claim 14.

16. A method for analysis of M. tuberculosis complex or Mycobacterium genus using the composition of claim 14, comprising:

(1) mixing the composition of claim 14 with bacterial DNA extracted from a sample of interest to prepare a gene analysis sample;

(3) quantifying the PCR product of Step (2) to obtain a quantitative curve; and

17. An rpoB-specific nucleic acid of Mycobacterium genus, wherein the nucleic acid is at least one selected from the group consisting of base sequences of the following SEQ ID NOs: 8 to 27:

M. acapulsensis-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 8; Mflavescens-speci&c nucleic acid containing a base sequence as set forth in SEQ ID NO:

9;

M. gαs/zn-specific nucleic acid containing a base sequence as set forth in SEQ ID NO: 10;

M intracellulare-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 11;

M kansasii-sped&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 12;

M pulveris-speάfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 13; M simiae-spocific nucleic acid containing a base sequence as set forth in SEQ ID NO: 14;

M. xenopi Schwabacher-speάfic nucleic acid containing a base sequence as set forth in SEQIDNO: 15;

M austroqfricanum-speάfic nucleic acid containing a base sequence as set forth in SEQ IDNO: 16; M. celatum-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO:

17;

M gastri-speύ&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 18; M gordbnαe-specific nucleic acid containing a base sequence as set forth in SEQ ID NO: 19;

M αgri-specific nucleic acid containing a base sequence as set forth in SEQ ID NO: 20;

M. asiaticum-specϊήc nucleic acid containing a base sequence as set forth in SEQ ID NO: 21;

M cekatum-spectfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 22;

M. diernhoferi-speάfic nucleic acid containing a base sequence as set forth in SEQ ID NO: 23; M fortuitum-speάSc nucleic acid containing a base sequence as set forth in SEQ ID NO:

24;

M nonchromogenicum-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 25;

M phlei-speά&c nucleic acid containing a base sequence as set forth in SEQ ID NO: 26; and

M. ge«αve«rø-specific nucleic acid containing a base sequence as set forth in SEQ ID NO: 27.