[DESCRIPTION]
[Invention Title]
COMPOSITION FOR DETECTION OF M. TUBERCULOSIS COMPLEX AND MYCOBACTERIA GENUS AND SIMULTANEOUS DETECTION METHOD FOR M. TUBERCULOSIS COMPLEX AND MYCOBACTERIA GENUS WITH MULTIPLEX REAL-TIME PCR USING THE SAME
[Technical Field]
The present invention relates to a composition for distinguishably detecting M. tuberculosis complex and mycobacteria genus and simultaneous analysis of M. tuberculosis and mycobacteria genus through real-time multiplex polymerase chain reaction (RT PCR) using the same. More specifically, the present invention relates to a composition for detecting M. tuberculosis and mycobacteria genus comprising (i) a primer and/or probe that targets an M. tuberculosis-specific gene, IS6110, (ii) a primer and/or probe that targets a mycobacteria genus-specific gene, inter-transcriptional spacer (ITS), and, optionally, (iii) a primer and/or probe that targets a plant-derived gene as an internal control.
[Background Art]
Tuberculosis is an infectious disease that exhibits the highest mortality rate in the history of mankind, which is caused by strains of mycobacteria genus having a thickness of 0.2 to 0.5 pm, a length of 1 to 4 pm and a rod shape.
Mycobacteria genus includes various strains of mycobacteria that infect a variety of humans and animals and result in respiratory diseases such as tuberculosis and diseases

such as leprosy and about 100 species are known to date (Shinnick TM et al., Mycobacterial taxonomy. Eur. J. Clin. Microbiol. Infect Dis. 1994;13(11):884-901).
As mycobacterium tuberculosis known as the most important mycobacterium that causes tuberculosis in humans, there are mycobacterium tuberculosis, mycobacterium bovis that rarely appears and the like. Mycobacterium leprae is known to cause leprosy (Shinnick, T.M. and Good, R.C. Mycobacterial taxonomy. Eur. J. Clin. Microbiol. Infect. Dis. 1994;13(11):884-901).
Tuberculosis is a disease prevalent in underdeveloped countries, 120,000 new cases of M tuberculosis are encountered annually in Korea and new tuberculosis infections reported and counted in 2003 was 30,687 (corresponding to 64 per one hundred thousand people), and Korea has a stigma of ranking top in the tuberculosis mortality among 30 OECD member countries (2004, statistical data from National Statistical Office).
Recently, acquired immune deficiency syndrome (AIDS) and other immune deficiency syndrome patients, infants having weaker immune systems and the like are infected with nontuberculous mycobacteria (NTM) and atypical tuberculosis patients that exhibit clinical symptoms similar to symptoms associated with M. tuberculosis infection are on the rise.
Examples of NTM include non-tuberculosis mycobacteria such as mycobacterium avium (M. avium-intracellulare or M. avium complex)^ M. fortuitum, M. chelonae, M. gordonae, M. szulagai, M. kansasii, M. genavense (Barnes, P.F. et al., Tuberculosis in patients with human immunodeficiency virus infection. N. Engl. J. Med. 1991;324(23): 1644-1650).
A number of species of these NTMs exhibit multidrug-resistance to M. tuberculosis drugs and have a difficulty in treatment (Kam, K.M. 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).
Mycobacterium avium complex (MAC) that is most commonly found in NTM is known to exhibit 10 to 100 times lower efficacy on primary tuberculosis drugs than M. tuberculosis and the American Thoracic Society (ATS) has suggested guidelines associated with diagnosis and treatment of diseases caused by diseases of respective species of nontuberculous mycobacteria (American Thoracic Society, Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med. 1997;156(2):S1-S25).
Accordingly, nontuberculous mycobacteria have considerably similar symptoms to tuberculous mycobacteria, but they may be different from tuberculous mycobacteria in terms of drugs for treatment. In order to suitably treat tuberculous mycobacteria and nontuberculous mycobacteria, respectively, accurately distinguishing tuberculous mycobacteria from nontuberculous mycobacteria and early detection and diagnoses thereof are required.
Generally methods for diagnosing tuberculosis include clinical symptom testing of patients, tuberculin skin response testing, X-ray imaging, M. tuberculosis testing and the like. Tuberculin testing which is the easiest method is simply to perform, but often exhibits false negatives in the case of anergy caused by serious tuberculosis, measles and immunosuppression. About 25% of X-ray images may be varied according to the ability of a detector and diagnosis using X-ray images thus depends on the ability of a detector of finding abnormal shadow and the ability of a detector of accurately judging the same.
Detection of M. tuberculosis is the most accurate method for diagnosing tuberculosis and includes smear testing, culture testing, molecular diagnosis testing and the like.

Smear testing predominantly uses staining in which acid fastness is confirmed through Ziehl-Neelsen staining. This method enables results to be obtained simply and rapidly, but does not enable tuberculous mycobacteria to be distinguished from nontuberculous mycobacteria and has low sensitivity.
Culture testing has high sensitivity to an extent that bacteria are detectable even if only about 10 bacteria are present in 1 ml of specimen and enables separation and accurate diagnosis of tuberculous mycobacteria. This method requires a long period of time, for example, culturing and observation for about 4 to 8 weeks by a well-trained researcher, thus being disadvantageous^ unsuitable for treatment.
BACTEC (Becton Dickinson, US) is a newly developed culture method in which bacillus is inoculated to a liquid culture medium containing C14 palmitate and the amount of 14CC>2 generated by metabolism of bacillus is estimated from growth quotient calculated using a radioactive isotope. This method enables results to be obtained after 16 days on average, but disadvantageously requires facilities and experts to handle radioactive isotopes.
Polymerase chain reaction (PCR) followed electrophoresis is a method in which presence of M. tuberculosis can be rapidly and accurately confirmed by amplifying a specific gene site within 2 to 3 hours. This method can detect M. tuberculosis from clinical specimens at a sensitivity and a specificity of 95% or more within a day and is thus useful (Wilson, S.M. 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, this method disadvantageously has a high risk of carry-over contamination and requires professional experts (Noordhoek, G.T. 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).

Meanwhile, in tuberculosis diagnosis, PCR testing has considerably high sensitivity and specificity in smear-positive specimens and the fact that acid-fast bacterium smear-positive and PCR negative specimens may be considered to be infected with NTM should be considered.
The U.S., which has a high frequency of NTM lung diseases, recommends that acid-fast bacterium smear-positive and PCR positive subjects be tentatively diagnosed with tuberculosis, acid-fast bacterium smear-positive and PCR-negative subjects be tentatively diagnosed with NTM, when whether or not PCR inhibitor is present is confirmed and the subjects tested negative again. It takes a culture time of 4 to 8 weeks to complete final NTM infection diagnosis (Centers for Disease Control and Prevention (CDC). Update: Nucleic acid amplification tests for tuberculosis. MMWR Morb. Mortal. Wkly. Rep. 2000; 49:593-4). Recently, NTM infection may be detected according to these guidelines, but, if possible, direct detection of NTM will be clinically further useful.
In Korea, onset rate of tuberculosis is high and a frequency of NTM diseases is low. Accordingly, acid-fast bacterium smear-positive subjects are generally considered to be infected with tuberculosis and are generally subjected to anti-tuberculosis drug treatment. However, recently, in Korea, identification rate of NTM and NTM diseases has increased, NTM may cause diseases hi those who have weak immune function, is not easy to diagnose, is difficult to treat due to high rates of drug resistance, and has a high recurrence rate (Scientific committee in Korean academy of tuberculosis and respiratory disease. National survey of mycobacterial diseases other than tuberculosis in Korea. Tuberc. Respir. Dis. 1995;42:277-94.). Accordingly, there is an increasing need for methods for distinguishably diagnosing NTM.

Recent methods for detecting NTM include AFB staining, methods generally used for distinguishing species of mycobacteria using a molecular biological method include PCR-RFLP using restriction enzyme and PCR-hybridization using specific probes.
All of these methods have low sensitivity and disadvantageously require prior culturing or entail complicated experimental steps, thus being unsuitable for use in rapid detection of M tuberculosis and mycobacteria genus at the early stage.
[Disclosure] [Technical Problem]
Therefore, the present invention has been made to solve the above problems and other technical problems that have yet to be resolved.
It is one object of the present invention to provide a primer and/or probe that has excellent sensitivity to IS6110, a gene site specific to M. tuberculosis, to accurately diagnose M. tuberculosis.
It is another object of the present invention to provide a primer and/or probe that detects a gene site highly specific to an ITS gene that is commonly present in mycobacteria genus and thus has excellent sensitivity, to confirm presence of mycobacteria genus (nontuberculous mycobacteria)
It is another object of the present invention to provide a method for simultaneously detecting M. tuberculosis complex and nontuberculous mycobacteria through real-time multiplex polymerase chain reaction using the primer and/or probe.
[Technical Solution]
h Primer for detection of M. tuberculosis or mycobacteria genus

The present invention relates to a composition for distinguishably detecting tuberculosis mycobacteria and nontuberculosis mycobacteria comprising: a primer containing one or more of a Seq. No. 1 base sequence and a Seq. No. 2 base sequence; and a primer containing one or more of Seq. Nos. 3 to 11 base sequences.
Preferably, the present invention relates to a composition for distinguishably detecting tuberculosis mycobacteria and nontuberculosis mycobacteria comprising: both a primer containing a Seq. No. 1 base sequence and a primer containing a Seq. No. 2 base sequence; or both a primer containing one or two or more of Seq. Nos. 3 to 9 base sequences and a primer containing one or two or more of Seq. Nos. 10 to 11 base sequences.
More preferably, the present invention relates to a composition for distinguishably detecting tuberculosis mycobacteria and nontuberculosis mycobacteria: comprising a primer containing a Seq. No. 1 base sequence; a primer containing one or two or more of Seq. Nos. 3 to 9 base sequences; and a primer containing one or two or more of Seq. Nos. 10 to 11 base sequences.
As used herein, the term "primer containing a Seq. No. x base sequence" includes a base sequence having a sequence homogeny of 95% or more. For example, in a case in which a primer is 20 bp, when two or more of 20 bp are different, a decrease in melting temperature is 5 degrees or higher, thus obtaining bad results, while, in a case in which only one thereof is different, when test is carried out at a slightly decreased annealing temperature during PCR, identical results can be obtained.
In this specification, hereinafter, "primer containing Seq. No. x base sequence" will simply be referred to as "Seq. No. x primer" for better illustration.
In this specification, the Seq. No. 1 primer and Seq. No. 2 primer are primers that target IS6110 gene sites, gene sites specific to M. tuberculosis complex.

Generally, "M tuberculosis" narrowly means mycobacterium tuberculosis. In the present invention, "M tuberculosis" broadly means mycobacterium tuberculosis as well as mycobacterium bovis, mycobacterium microti, and mycobacterium africanum. In some cases, this is referred to as "M tuberculosis complex" or "TB complex".
The IS6110 gene is an insertion sequence which is found in M. tuberculosis complex such as mycobacterium tuberculosis and mycobacteria 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., J. Clin. Microbiol. 1990;28(12):2668 -2673). However, Kent et al., reported that IS6110 exhibits a difference in false positive risk ratio depending on the selection position of primer (J. Clin. Microbiol. 1995;33(9):229G-2293).
Accordingly, the inventors of the present invention researched sites having low false positive risk through analysis of base sequence. As a result, as can be seen from the following examples, the Seq. No. 1 primer and Seq. No. 2 primer are primer base sequences that amplify only the IS6110 gene of M. tuberculosis complex and exhibit a considerably high sensitivity of 97% or more and a positive predictive ratio of 99% or more in IS6110 gene site of M. tuberculosis complex, which means that the risk of false positives is considerably low. Such a primer that exhibits a high sensitivity and a positive predictive ratio has been not found to date.
Accordingly, when the Seq. No. 1 primer, the Seq. No. 2 primer or a combination thereof is used, M. tuberculosis complex can be detected with high reliability.
In order to confirm the presence/absence of nontuberculosis mycobacteria, identification of mycobacteria is important. 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 Laid-open No. 10-2001-0038701).
However, this patent uses PCR-RFLP and thus has problems such as the necessity of prior culturing due to low sensitivity and complicated experimental steps, thus being unsuitable for use in rapid detection of M. tuberculosis complex and mycobacteria genus at the early stage.
Accordingly, in order to solve these problems, the present inventors developed a novel primer specific to rpoB gene sites that forms a PCR amplification product of 100 bp or less that is suitable for real-time PCR (Kang Jin Seok et al., Korean Patent Application No. 2008-0090156).
However, rpoB is a gene that encodes for an RNA polymerase B-subunit, and it can be seen from specificity testing results of this application that various species of microorganisms do not exhibit positives, but Nocardia genus microorganisms that exhibit symptoms of pneumonia similar to tuberculosis and rhodococcus genus microorganisms similar to mycobacteria genus exhibit positives due to presence of similar base sequences that perform common actions in some microorganisms.
Meanwhile, in order to confirm presence of nontuberculosis mycobacteria, identification of mycobacteria genus is important. It is known that an ITS gene as well as an rpoB gene are contained in the mycobacteria genus and are thus used for identification of bacterial species (Kim In Soo et al., Korean Patent No. 0725579).
However, this patent uses a primer that forms a PCR product with a size of about 250 to 450 bp for PCR-reverse hybridization and is thus disadvantageous^ unsuitable for real-time PCR for rapid detection.
Accordingly, the present inventors developed primers that form amplification products having a size suitable for real-time PCR in ITS sites. That is, the Seq. No. 3 to

11 primers are primers that target ITS gene sites, gene sites specific to mycobacteria genus. These primers form PCR amplification products with a size of 100 bp or less. Accordingly, these primers advantageously enable mycobacteria genus to be considerably reliably and rapidly detected and exhibit a higher specificity than before since they do not react with Nocardia genus microorganisms and rhodococcus genus microorganisms.
The composition for detecting M. tuberculosis or mycobacteria genus according to the present invention may further selectively comprise an internal control primer. This internal control is used to confirm whether or not a false negative problem has occurred, that is, PCR reaction is normally performed. Regardless of presence of M tuberculosis or mycobacteria in the sample, normally expressed genes may be randomly selected.
In a preferred embodiment, the internal control primer may be a plant-derived gene-specific primer added to all samples.
This plant-derived gene is preferably lectin and a lectin gene-specific primer may be a primer containing Seq. No. 15 base sequence or a primer containing a Seq. No. 16 base sequence.
In the present invention, base sequences of the Seq. Nos. 1 to 11 primers, the Seq. No. 15 primer and the Seq. No. 16 primer are given in Table 1 below.

ITS AS 1 G ATG CTC GCA ACC ACT ATC CA 21 10 91
ITS AS2 G ATG CTC GCA ACC ACT ATT CA 21 11 91
L554SN GCC TCT TGG TTG CTT CTT TG 20 15 148
L701AS TTC CCC AGG TAT GTC GAG TC 20 16 148
In the sequences above, "R" and "Y" are combined base sequences and exhibit
"R-A+G" and "Y=C+T", respectively.
As mentioned above, the present invention provides a composition for detecting M. tuberculosis comprising one or two or more of a sense primer containing a Seq. No. 1 base sequence and an antisense primer containing a Seq. No. 2 base sequence, as a novel primer specific to M. tuberculosis IS6110 gene.
In addition, the present invention provides a composition for detecting the mycobacteria genus comprising: one or two or more of sense primers containing Seq. Nos. 3 to 9 base sequences; one or two or more of antisense primers containing Seq. Nos. 10 to 11 base sequences; or a combination thereof, as a primer specific to the ITS gene of mycobacteria.
The primers specific to the ITS gene of the mycobacteria genus form PCR amplification products having a length of 100 bp or less that are suitable for real-time PCR.
In a preferred embodiment, the mycobacteria may be mycobacterium tuberculosis or mycobacterium bovis. Detection reliability of M tuberculosis can be further improved by analyzing an IS6110 gene of M, tuberculosis complex with the ITS gene.
In a preferred embodiment, the mycobacteria may be nontuberculosis mycobacteria (NTM). In this case, presence and type of nontuberculosis mycobacteria, rather than M, tuberculosis complex, can be advantageously confirmed through analysis of base sequence of ITS genes.

In addition, the present inventors confirmed that primers containing Seq. Nos. 3 to 11 base sequences are effective in identifying the following 42 species of mycobacteria and have high specificity.
2. Probe for detecting M. tuberculosis or mycobacteria genus
In another aspect, the present invention provides a composition for detecting M tuberculosis or mycobacteria genus, comprising one or two or more selected from the group consisting of: a probe containing a Seq. No. 12 base sequence; a probe containing a Seq. No. 13 base sequence; and a probe containing a Seq. No. 14 base sequence.
The composition may optionally further comprise an internal control probe and the internal control probe is preferably a probe that is specific to a plant-derived gene, lectin, and contains a Seq. No. 17 base sequence.
In the present invention, the term "probe containing a Seq. No. x base sequence" includes a base sequence having a sequence homogeny of 95% or more. In this

specification, hereinafter, "probe containing Seq. No. x base sequence" will be simply referred to as "Seq. No. x probe" for better illustration.
The Seq. No. 12 probe specifically reacts with the IS6110 gene site, and the Seq. No. 13 probe and the Seq. No. 14 probe are probes that commonly react with ITS genes of detectable all mycobacteria genus.
As such, whether or not mycobacteria in samples is M. tuberculosis can be more accurately detected through the probe specific to the IS6110 and ITS genes, and presence of mycobacteria can be confirmed by confirming presence of ITS genes.
In particular, the probe specific to IS6110 or ITS genes according to the present invention is useful for quantitative PCR assay using taqman assay or molecular beacon assay.
Accordingly, in a preferred embodiment, the probe are labeled with a fluorescent material at 5' and 3'-ends thereof, and a fluorescent material (reporter) labeled at the 5'-end and a fluorescent material (quencher) labeled at the 3'-end may interfere with each other. Accordingly, when a probe binds to IS6110 or ITS gene present in samples, color development of the probe is limited and when polymerase chain reaction is performed, the probe is decomposed and the fluorescent material labeled at the 5'-end thereof is quenched, while the fluorescent material labeled on the 3'-end undergoes color development.
The fluorescent material labeled at the 5'-end of the probe is not particularly limited and, for example, the fluorescent material may be selected from the group consisting of 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6-carboxyfluorescein, and Cyanine-5 (Cy5) and is not limited thereto.

In addition, the fluorescent material labeled at the 3'-end may be 6-carboxytetramethyl-rhodamine (TAMRA) or black hole quencher-1,2,3 (BHQ-1,2,3), but is not limited thereto.
In the present invention, base sequences of the Seq. Nos. 12 to 14 probes, and the Seq. No. 17 probe are given in Table 2 below.
3. Composition and kit for real-time PCR assay
The present invention provides a composition for distinguishing, detecting and analyzing M. tuberculosis and mycobacteria genus using real-time PCR assay, comprising: a primer containing one or two or more of a Seq. No. 1 base sequence and a Seq. No. 2 base sequence; a primer containing one or two or more of Seq. Nos. 3 to 11 base sequences; and a probe containing one or two or more of Seq. Nos. 12 to 14 base sequences.
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 Seq. No. 15 base sequence or an antisense primer containing a Seq. No. 16 base sequence, and the internal control probe may be a probe containing a Seq. No. 17 base sequence.

That is, the composition for quantitative analysis according to the present invention comprises the primer for detecting M. tuberculosis or nontuberculosis mycobacteria, or the probe for detecting nontuberculosis mycobacteria, and optionally the internal control primer and probe.
Real-time PCR assay in which two or three species of genes are amplified together with DNA extracted from clinical specimens in a single tube and the resulting products are real-time analyzed and detected can be carried out using the composition according to the present invention.
In a preferred embodiment, the composition comprises:
(i) a primer and probe mixture specific to an IS6110 gene of M. tuberculosis: comprising a sense primer containing a Seq. No. 1 base sequence and an antisense primer containing a Seq. No. 2 base sequence; and a probe containing a Seq. No. 12 base sequence;
(ii) a primer and probe mixture specific to an ITS gene of mycobacteria genus comprising: a sense primer containing Seq. Nos. 3 to 9 base sequences and an antisense primer containing Seq. Nos. 10 to 11 base sequences; and a probe containing a Seq. No. 13 base sequence and/or a probe containing a Seq. No. 14 base sequence;
(iii) a primer and probe mixture specific to an internal control gene comprising: a sense primer containing a Seq. No. 15 base sequence and an antisense primer containing a Seq. No. 16 base sequence; and a probe containing a Seq. No. 17 base sequence; and
(iv) a PCR reaction mixture comprising a buffer, DNA polymerase, dNTP and sterilized distilled water.
The composition for analysis comprises groups of primers and three groups of probes. The inventors of the present invention performed real-time multiplex PCR using the composition. As a result, the inventors confirmed that M. tuberculosis and

mycobacteria in specimens can be detected and diagnosed with substantially complete exclusion of the risk of false positivity.
Specifically, the mixture (i) can amplify only M. tuberculosis complex and comprises a primer that exhibits considerably high sensitivity to IS6110 gene sites of 97% or more and a positive predictive ratio thereto of 99% or more and a probe that specifically binds to IS6110 gene sites amplified using the primer.
The mixture (ii) can amplify a mycobacteria genus-specific gene, ITS gene and comprises a primer that forms an ITS gene site having a length suitable for analysis using real-time PCR and a probe specifically linked to the ITS gene site amplified using the primer.
At this time, the probe binds to ITS genes of detectable all mycobacteria genus and presence of mycobacteria can thus be confirmed through the binding.
If necessary, the composition may further comprise one or more probes that contain ITS gene sites having different base sequences extracted from different species of mycobacteria. In this case, the species of respective mycobacteria can be confirmed.
Accordingly, when an analysis kit comprising the composition is used, genes specific to tuberculosis can be quantitatively analyzed in a simple and accurate manner, presence of nontuberculous mycobacteria can be confirmed and the analysis kit can thus be widely used for accurate diagnosis of tuberculosis. In some cases, respective mixtures are separately analyzed and may be judged as a set.
Such real-time PCR assay may be carried out using a commercially available real-time PCR apparatus and examples of the real-time PCR apparatus include, but are not limited to, SLAN real-time PCR detection systems (LG Life Sciences, Korea), LightCycler™ (Roche, Germany), ABI PRISMTM 7000/7700 (Applied Biosystems,

USA), iCycler1M (Bio-Rad, USA), Rotor-Gene1M (Corbett, Australia), Opticon™ (PharmaTech, USA) and the like.
4. Method for analyzing M. tuberculosis or mycobacteria genus
The composition for analysis according to the present invention may be used for qualitative analysis to confirm presence of M tuberculosis or mycobacteria genus and for quantitative analysis thereof. That is, the present invention provides a method for quantitatively or qualitatively analyzing M. tuberculosis or nontuberculosis mycobacteria using the composition for analysis.
The qualitative analysis to confirm presence of M. tuberculosis or mycobacteria genus may be carried out by performing real-time PCR using the composition for analysis and confirming a point at which peaks are observed.
In addition, the quantitative analysis may be carried out through comparison with a standard curve. In a preferred embodiment, quantitative analysis may be carried out through the following steps using the composition for analysis:
(1) mixing a composition for analysis with bacterial DNA extracted from a sample to prepare a sample for gene analysis;
(2) subjecting the sample for gene analysis to real-time PCR to obtain a PCR product;
(3) quantifying the PCR product to obtain a quantitative curve; and
(4) quantifying IS6110-, ITS- and internal control- specific genes, present in the bacterial DNA extracted from the sample using the quantitative curve.
That is, when bacterial DNA extracted from a sample is added to the composition for quantitative analysis comprising groups of primer and three groups of probes, that is, primer pairs and probes specific to M. tuberculosis gene IS6110, primer pairs and probes

specific to an ITS gene of mycobacteria genus, and primer pairs and probes specific to internal control genes such as the lectin gene, and real-time PCR is performed, respective primer pairs and probes specific to the IS6110 gene, ITS gene and internal control gene such as lectin gene bind to DNA and PCR products are amplified.
In a case in which a taqman analysis is used as a method for analyzing PCR products, IS6110-, ITS- and internal control-specific genes can be analyzed by confirming amplification of genes using a phenomenon in which probes linked to IS6110, ITS and lectin genes are decomposed and emit light.
[Brief description of the drawings]
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is an image showing real-time multiplex PCR results of an M. tuberculosis complex-positive sample using the composition for analysis according to the present invention;
FIG. 2 is an image showing real-time multiplex PCR results of mycobacteria genus-positive sample using the composition for analysis according to the present invention; and
FIG. 3 is an image showing real-time multiplex PCR results of a negative sample using the composition for analysis according to the present invention.
[Best mode]

Now, the present invention will be described in more detail with reference to the following examples. These examples should not be construed as limiting the scope and spirit of the present invention.
Example 1: Production of primers and probes
The fact that the IS6110-specific primers and probes used for the present invention were primer base sequences that can amplify only M. tuberculosis complex was confirmed by analyzing base sequences deposited under accession number NC000962 in GenBank (www.ncbi.nlm.nih.gov') managed by NCBI, a National institutes of Health (NIH)-affiliated organization using DNAsis produced by Hitachi Software Engineering Co., Ltd., determining the base sequence and analyzing the base sequence using BLAST (www.ncbi.nlm.nih.gov/BLAST/).
In addition, the fact that the ITS-specific primers and probes used for the present invention are primer base sequences that can amplify only mycobacteria genus were confirmed by obtaining a mycobacteria ITS gene from Genebank Entrez, analyzing a preservation site using clustal X, determining the base sequence and analyzing the base sequence using BLAST (www.ncbi.nlm.nih.gov/BLAST/).
Base sequences of the plant-derived gene-specific primers and probes are obtained from patents published in the art.
Example 2: Synthesis of primers
The primers analyzed in Example 1 were synthesized by Metabion Coiporation (Germany) using the method such as "Oligonucleotide Synthesis" described in a paragraph 10.42 of Molecular Cloning 3rd ed. (Sambrook and Rusell, Cold Spring Harbor Laboratory Press, New York, USA, 2001).

Example 3: Extraction of mycobacterial DNA from clinical specimen or standard strain culture medium
1 to 2 ml of sputum of a suspected tuberculosis patient or standard strain culture medium and 1 to 2 ml of 4N NaOH were placed in a 15 ml tube, following by sufficient stirring and centrifugation at 4,000 rpm for 20 minutes. A supernatant was removed and 10 ml of PBS buffer (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04, 2 mM KH2P04) was added to the resulting precipitate, followed by sufficiently stirring and centrifuging at 4,000 rpm for 20 minutes. The supernatant was discarded and the resulting precipitate was transferred to a 1.5 ml tube and 1 ml of PBS buffer was added thereto, followed by stirring and centrifugation at 13,000 rpm for 5 minutes. The supernatant was discarded again and 50-100 ui of a 5% (w/v) Chelex 100 resin (Bio-Rad Corp.) was added to the resulting precipitate, the mixture was heated at 100°C for 20 minutes and centrifuged at 13,000 rpm for 3 minutes and the separated DNA supernatant was used as template DNA for PCR.
Example 4: Real-time multiplex PCR reaction analysis using primers and probes
PCR reaction compositions were prepared according to a PCR reaction composition formula shown in the following Table 3 and PCR reaction was performed in a SLAN real-time PCR detection system (LG Life Sciences Ltd., Korea) under reaction conditions shown in the following Table 4.
Reaction products were measured in real-time and results were analyzed using a SLAN 7.0 program after reaction was completed.
It was determined as follows: FIG. 1: M. tuberculosis complex positive, FIG. 2: Mycobacteria genus-positive and FIG. 3: Negative.

Example 6: Comparison tests of diagnosis effects of M. tuberculosis and mycobacteria genus using a conventional culture method (MGIT, BD Corp., USA) and the kit of the present invention
Diagnosis effects of M. tuberculosis and mycobacteria genus were compared between a conventional culture method (MGIT, BD Corp., US) and the kit of the present invention.
The culture method used for comparison was carried out in accordance with according to the manufacturer's instructions and the species of cultured positive specimens were confirmed using an amplicor MTB kit (Roche-diagnosis, USA) and a base sequence analysis. The results are shown in Table 5.
[Table 5] Comparison test results
(1) Result table

As used herein, the term "sensitivity" refers to the proportion of people with disease in whom the test result is positive, that is, the proportion in which people who suffer from a disease are judged to have a disease. As used herein, the term "specificity" refers to the proportion in which healthy people are detected to be negative, that is, the proportion in which normal people are judged to be normal. The term "positive predictive rate" refers to the probability in which people who are considered to be positive through a diagnosis method has the corresponding disease. The term "negative predictive rate" refers to the probability in which people who are detected to be positive through a diagnosis method does not have the corresponding disease.
According to the results of Table 5 above, 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 mycobacteria and nontuberculous mycobacteria. In particular, the sensitivity to M. tuberculosis was 97% or more and the specificity was 99%, which indicates that M. tuberculosis can be accurately identified.
Example 7: Comparison test of diagnosis effects between nocardia genus microorganisms exhibiting symptoms of pneumonia similar to tuberculosis and rhodococcus genus microorganisms similar to mycobacteria genus
With respect to nocardia genus microorganisms that may exhibit similar symptoms to tuberculosis and rhodococcus genus microorganisms, the results obtained from use of the kit according to Korean Patent Application No. 2008-0090156 were

compared with the results obtained by RT-PCR using the kit according to the present invention and are shown in Table 6.
As can be seen from Table 6, the present invention completely eliminates false positive probability associated with conventional patent compositions.
24

It can be seen from the afore-mentioned results that NTM can be detected at a considerably high specificity by using the composition of the present invention, as compared to patents in the related art.
Example 8: RT-PCR results of the present invention in various mycobacteria 42 species of mycobacteria were subjected to RT-PCR in accordance with the method of Examples 3 and 4 and the results are shown in the following Table 7.

(In Table 7, when a mycobacterium has a value lower than 35, the mycobacterium is defined as "positive" and IC means internal control. In addition, Ct is a threshold cycle, which means a number of cycles that passes a critical value as a result of RT-PCR and No Ct means absence of a threshold cycle.)
As can be seen from Table 7, as a result of RT-PCR according to the present invention, all of M. tuberculosis H37Rv, M. bovis, M. bovis BCG, M. africanum, and M. microti that belong to the M. tuberculosis complex tested M. tuberculosis positive and the remaining mycobacteria tested mycobacteria positive. Accordingly, RT-PCR using the analysis composition according to the present invention is useful for detection of M. tuberculosis and mycobacteria genus.
[Industrial applicability]
As apparent from the afore-going, when real-time PCR is performed using the composition for analysis according to the present invention, detection of M. tuberculosis and mycobacteria genus can be realized at a high sensitivity and specificity within a short period of time, as compared to conventional methods.
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] [Claim 1]
A composition for distinguishably detecting tuberculosis mycobacteria and nontuberculosis mycobacteria comprising:
a primer containing one or more of a Seq. No. 1 base sequence and a Seq. No. 2 base sequence; and
a primer containing one or more of Seq. Nos. 3 to 11 base sequences.
[Claim 2]
The composition according to claim 1, wherein the composition comprises: both a primer containing a Seq. No. 1 base sequence; and a primer containing a Seq. No. 2 base sequence.
[Claim 31
The composition according to claim 1, wherein the composition comprises: both a primer containing one or two or more of Seq. Nos. 3 to 9 base sequences; and a primer containing one or two or more of Seq. Nos. 10 to 11 base sequences.
[Claim 4]
The composition according to claim 1, wherein the composition comprises:
a primer containing a Seq. No. 1 base sequence;
a primer containing a Seq. No. 2 base sequence;
a primer containing one or two or more of Seq. Nos. 3 to 9 base sequences; and
a primer containing one or two or more of Seq. Nos. 10 to 11 base sequences.

[Claim 51
The composition according to any one of claims 1 to 4, further comprising an internal control primer.
[Claim 6]
The composition according to claim 5, wherein the internal control primer is a plant-derived gene-specific primer.
[Claim 7]
The composition according to claim 6, wherein the plant-derived gene is lectin and the plant-derived gene-specific primer comprises one or two or more of a primer containing a Seq. No. 15 base sequence and a primer containing a Seq. No. 16 base sequence.
[Claim 8]
A composition for detecting M. tuberculosis comprising one or two or more of a sense primer containing a Seq. No. 1 base sequence and an antisense primer containing a Seq. No. 2 base sequence, as a primer specific to an IS6110 gene of M. tuberculosis.
[Claim 9]
A composition for detecting the mycobacteria genus comprising one or two or more of sense primers containing Seq. Nos. 3 to 9 base sequences; one or two or more of antisense primers containing Seq. Nos. 10 to 11 base sequences; or a combination thereof, as a primer specific to an ITS gene of the mycobacteria genus.

[Claim 10]
The composition according to claim 9, wherein the primer specific to an ITS gene of the mycobacteria genus forms a PCR amplification product with a length of 100 bp or less.
[Claim 11]
A composition for detecting M. tuberculosis or mycobacteria genus, comprising one or two or more selected from the group consisting of: a probe containing a Seq. No. 12 base sequence; a probe containing a Seq. No. 13 base sequence; and a probe containing a Seq. No. 14 base sequence.
[Claim 12]
The composition according to claim 11, wherein the probe are labeled with a fluorescent material at the 5'-end and the 3'-end thereof and the fluorescent material (reporter) labeled at the 5'-end and the fluorescent material (quencher) labeled on the 3'-end interfere with each other.
[Claim 13]
The composition according to claim 12, wherein the fluorescent material labeled on the 5'-end is selected firom the group consisting of 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6-carboxyfluorescein, and Cyanine-5 (Cy5) and the fluorescent material labeled on the 3'-end is 6-carboxytetramethyl-rhodamine (TAMRA) or black hole quencher-1,2,3 (BHQ-1,2,3).
[Claim 14]

A composition for distinguishing, detecting and analyzing M. tuberculosis and mycobacteria genus using real-time PCR assay comprising:
a primer containing one or two or more of a Seq. No. 1 base sequence and a Seq. No. 2 base sequence;
a primer containing one or two or more of Seq. Nos. 3 to 11 base sequences; and
a probe containing one or two or more of Seq. Nos. 12 to 14 base sequences.
[Claim 15]
The composition according to claim 14, further comprising: an internal control primer; and an internal control probe.
[Claim 16]
The composition according to claim 15, wherein the internal control primer is a sense primer containing a Seq. No. 15 base sequence or an antisense primer containing a Seq. No. 16 base sequence, and the internal control probe is a probe containing a Seq. No. 17 base sequence.
[Claim 17]
The composition according to claim 14, wherein the composition comprises;
(i) a primer and probe mixture specific to an IS6110 gene of M. tuberculosis, comprising: a sense primer containing a Seq. No. 1 base sequence and an antisense primer containing a Seq. No. 2 base sequence; and a probe containing a Seq. No. 12 base sequence;

(ii) a primer and probe mixture specific to an ITS gene of mycobacteria genus, comprising: a sense primer containing Seq. Nos. 3 to 9 base sequences and an antisense primer containing Seq. Nos. 10 to 11 base sequences; and a probe containing a Seq. No. 13 base sequence and/or a probe containing a Seq. No. 14 base sequence;
(iii) a primer and probe mixture specific to an internal control gene, comprising: a sense primer containing a Seq. No. 15 base sequence and an antisense primer containing Seq. No. 16 base sequence; and a probe containing a Seq. No. 17 base sequence; and
(iv) a PCR reaction mixture comprising a buffer, DNA polymerase, dNTP and sterilized distilled water.
[Claim 181
An M. tuberculosis' analysis kit comprising the composition for analysis according to claim 17.
[Claim 19]
A method for analyzing M. tuberculosis or mycobacteria genus using the composition according to claim 17, comprising:
(1) mixing a composition for analysis with bacterial DNA extracted from a sample to prepare a sample for gene analysis;
(2) subjecting the sample for gene analysis to real-time PCR to obtain a PCR product;
(3) quantifying the PCR product to obtain a quantitative curve; and
(4) quantifying IS6110-, ITS- and internal control- specific genes, present in the bacterial DNA extracted from the sample using the quantitative curve.

[Claim 20]
An ITS gene-specific primer comprising one of Seq. Nos. 3 to 11 base sequences.