KR20100031188A

KR20100031188A - 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: KR20100031188A
Application number: KR1020080090156A
Authority: KR
Inventors: 강진석; 박영석; 이재성
Original assignee: 주식회사 엘지생명과학
Priority date: 2008-09-12
Filing date: 2008-09-12
Publication date: 2010-03-22

Abstract

PURPOSE: A composition for detecting M. Tuberculosis TB complex or Mycobacteria genus is provided to simultaneously detect M. Tuberculosis TB complex or Mycobacteria genus through real time PCR. CONSTITUTION: A composition for detecting M. Tuberculosis TB complex or Mycobacteria genus comprises: one or more primer selected from a primer containing sequence number 1, 2, 3, and 4, respectively. The composition further contains an internal control primer. A composition for detecting M. Tuberculosis TB complex contains a sense primer containing a sequence of sequence number 1 and/or antisense primer containing a sequence of sequence number 2 as a specific primer to IS6110 gene. A composition for detecting Mycobacteria genus contains a sense primer containing a sequence of sequence number 3 and/or antisense primer containing a sequence of sequence number 4.

Description

Composition for detection of Mycobacterium tuberculosis group or Mycobacteria genus and method for simultaneous analysis of Mycobacterium tuberculosis and Mycobacteria genus by real-time multiplex polymerase chain reaction using the same Mycobacteria Genus with Multiplex Real Time PCR Using the Same}

The present invention relates to a composition for detecting Mycobacterium tuberculosis group or Mycobacteria genus and a method for simultaneous analysis of Mycobacterium tuberculosis and Mycobacteria genus by real-time multiplex polymerase chain reaction using the same. Primers and / or probes, (ii) primers and / or probes targeting rpoB, a gene specific for mycobacteria, and optionally (iii) primers targeting plant-derived genes as internal controls. And / or relates to a composition for detecting the genus Mycobacterium or Mycobacteria comprising a probe.

Tuberculosis is the most life-threatening epidemic in human history, caused by Mycobacteria genus tuberculosis, which is 0.2-0.5 μm thick and 1-4 μm long.

The Mycobacteria Genus includes a number of species that infect humans and animals and cause diseases such as respiratory diseases such as tuberculosis and leprosy, and about 100 or more species are known so far (Shinnick TM et al., Mycobacterial). taxonomy.Eur J Clin Microbiol Infect Dis. 1994; 13 (11): 884-901).

The most important causes of tuberculosis in humans are Mycobacterium tuberculosis and the rare mycobacterium bovis. Mycobacterium leprae is known to cause leprosy (Shinnick). TM and Good RC.Mycobacterial taxonomy.Eur J Clin Microbiol Infect Dis. 1994; 13 (11): 884-901).

The tuberculosis is one of the diseases of the less developed countries, and 120,000 new cases of tuberculosis infections still occur in Korea every year, and the number of newly diagnosed tuberculosis patients reported in 2003 reaches 30,687 (64 per 100,000 population) and tuberculosis among 30 OECD countries. It has the number one stigma of mortality rate (Statistical Statistics of 2004).

Recently, there has been a steady increase in the number of patients with similar tuberculosis, which has been infected by patients with acquired immunodeficiency syndrome (AIDS) and other immunodeficiency disorders or infants with weak immunity due to nontuberculous mycobacteria (NTM).

These NTMs include M. avium-intracellulare or M. avium complex, Mycobacterium potuitum, Mycobacterium kerone, and Mycobacterium Gordone. (M. gordonae), M. szulagai, M. kansasii, M. africanum, Mycobacterium jennavens Non-tuberculosis mycobacteria such as M. genavense) (Barnes PF et al., Tuberculosis in patients with human immunodeficiency virusinfection. N Engl J Med. 1991; 324 (23): 1644-1650).

Many of these NTMs have multiple resistance to M. tuberculosis treatment agents, making them difficult to treat (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, in the case of the group of tuberculosis bacteria (MAC), which is most frequently found in NTM, the effect on the first tuberculosis drug is 10 to 100 times less than that of tuberculosis bacteria. Guidelines for treatment and treatment (American Thoracic Society, Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med. 1997; 156 (2): S1-S25).

Therefore, while their symptoms are very similar to tuberculosis, the treatment agents may be completely different, so it is essential to accurately distinguish tuberculosis and non-tuberculosis early and differential diagnosis in order to perform appropriate treatment in response to each of them. It is requested.

Common methods for diagnosing tuberculosis include clinical symptoms of the patient, tuberculin skin tests, X-rays and tuberculosis bacteria. The simplest method, the tuberculin test, is simple to perform, but is often false negative in severe tuberculosis, measles and immunosuppression, and x-rays vary by about 25% depending on the reader's ability. The diagnosis depends on the reader's ability to detect the abnormal shadows and to accurately determine the abnormal shadows.

The detection of tuberculosis bacteria is a reliable method for diagnosing tuberculosis, including smear, culture and molecular diagnostic tests.

Smear staining is commonly used to detect acidity by Ziehl-Neelsen staining.However, the results can be obtained quickly and easily, but it is not possible to distinguish between tuberculosis and non-tuberculosis. But the sensitivity is also low.

The culture test method is highly sensitive enough to detect bacteria even if only 10 bacteria are present in 1 ml of the sample, and can accurately diagnose tuberculosis bacteria, but it should be observed for 4-8 weeks by well trained personnel. It has a very long time and is not suitable for treatment.

The BACTEC method (Becton Dickinson, USA), developed as a new culture method, inoculates bacteria into a liquid medium containing C14 palmitate and converts ¹⁴ CO ₂ from metabolism of bacteria into a growth index using radioisotopes. It is a technique to show and judge, but the result can be obtained in an average of about 16 days, but there is a problem of having a facility and a professional manpower to handle the radioisotope.

Polymerase chain reaction (PCR) method using electrophoresis is a method that can quickly and accurately determine the presence of Mycobacterium tuberculosis by amplifying a specific gene region in 2-3 hours. It can be useful for detecting tuberculosis bacteria from clinical specimens with sensitivity and specificity of more than 95% within a 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, carry-over contamination is susceptible to problems and requires specialized personnel (Noordhoek GT etal., 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).

On the other hand, in the diagnosis of tuberculosis, the PCR test has high sensitivity and specificity, especially in smear-positive specimens, and if it is anti-bacterial smear-positive and PCR-negative, the possibility of NTM should be considered.

In the United States, where the frequency of NTM pulmonary disease is high, it is recommended that a positive anti-bacterial smear is diagnosed as pulmonary tuberculosis if the PCR is positive, and if it is negative, if there is a PCR inhibitor, it is recommended to be temporarily infected with NTM. A 4-8 week incubation period is required to diagnose the final NTM infection (Centers for Disease Control and Prevention (CDC). Update: Nucleic acid amplification tests for tuberculosis.MMWR Morb Mortal Wkly Rep 2000; 49: 593-4). Currently, these guidelines can be used to estimate NTM infection, but it would be more clinically useful to be able to identify NTM directly.

In Korea, the high prevalence of tuberculosis and the incidence of NTM disease are low, and most of them are considered to be tuberculosis-resistant and anti-tuberculosis drugs are treated. Recently, however, NTM isolation and NTM disease have increased in Korea, and NTM can cause disease in immunocompromised patients, is not easy to diagnose, and is difficult to treat due to high drug resistance and 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.). Therefore, the need for diagnostic methods to distinguish NTM is increasing.

Currently NTM detection method is AFB staining method, commonly used to distinguish the species of mycobacteria using molecular biological techniques, PCR-RFLP method using a restriction enzyme and PCR using a specific probe There is a hybridization method.

All of the above methods have low sensitivity and are not suitable for early detection of Mycobacterium tuberculosis and Mycobacteria due to problems such as culturing first or complex experimental steps.

It is an object of the present invention to provide primers and / or probes with excellent sensitivity to the IS6110 gene, a Mycobacterium tuberculosis specific gene site, for accurate diagnosis of Mycobacterium tuberculosis.

In addition, an object of the present invention is to provide a primer and / or probe having excellent sensitivity by detecting a specific specific gene region in rpoB gene commonly present in mycobacteria, to detect the presence of the mycobacteria genus.

Still another object of the present invention is to provide a method for simultaneously detecting tuberculosis bacteria and non-tuberculosis bacteria by real-time multiplex polymerase chain reaction using the primers and / or probes.

One. Primer for the detection of Mycobacterium tuberculosis or Mycobacteria

The present invention is a primer comprising a nucleotide sequence of SEQ ID NO: 1; A primer comprising a nucleotide sequence of SEQ ID NO: 2; A primer comprising a nucleotide sequence of SEQ ID NO: 3; And a primer comprising a nucleotide sequence of SEQ ID NO: 4; and a composition for detecting Mycobacterium tuberculosis or non-tuberculosis mycobacteria comprising one or two or more primers selected from the group consisting of:

In the present invention, the "primer including the nucleotide sequence of SEQ ID NO: x" is a concept including a nucleotide sequence each having a sequence homology of 95% or more. For example, if the primer is 20bp, if two or more of the 20bps are different, the decrease in the melting temperature will be 5 degrees or more. However, if only one is incorrect, the annealing temperature will be changed during PCR. If you experiment a little down, you get the same result.

In the present specification, for the convenience of description, the term "primer including the nucleotide sequence of SEQ ID NO: x" is abbreviated as "primary primer of the sequence number x".

In the present invention, the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 are primers that target the IS6110 gene site, which is a gene site specific to Mycobacterium tuberculosis group.

Generally, 'mycobacterium tuberculosis' refers to Mycobacterium tuberculosis, which is a narrow tuberculosis bacterium, but the present invention includes Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis and Mycobacterium africanum, in addition to human tuberculosis Mycobacterium tuberculosis. It is expressed as 'TB group' or 'TB complex'.

The IS6110 gene is an insertion sequence present in Mycobacterium tuberculosis group, such as Mycobacterium tuberculosis and Mycobacterium tuberculosis, Mycobacterium tuberculosis. It is known that 10-12 copies are present in Mycobacterium tuberculosis group. Mainly used (thierry D et al., J Clin Microbiol. 1990; 28 (12): 2668-2673). However, KENT et al. Reported that there is a difference in false positive risk according to the primer selection position for IS6110 (J. Clin Microbiol 1995; 33 (9): 2290-2293).

Thus, the present inventors have studied the site of the risk of false positives through sequencing, as can be seen in the following examples, the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 is only the IS6110 gene of Mycobacterium tuberculosis group It was confirmed that the risk of false positives was extremely low because it showed a primer sequence that could be amplified and showed very high sensitivity of 97% or more and a positive predictive value of 99% or more in the IS6110 gene region of the tuberculosis group. Primers that exhibit high sensitivity and positive predictive rates have not been identified to date.

Therefore, when using the primer of SEQ ID NO: 1, the primer of SEQ ID NO: 2, or both, there is an advantage that the tuberculosis group can be detected very reliably.

On the other hand, Doucet-populaire et al. Reported that about 7% of non-tuberculosis mycobacteria carry a partial or analogous IS6110 gene that poses a risk of false positives (Tuber Lung Dis 1996; 77 (4): 358-62).

Therefore, in order to confirm the existence of non-tuberculosis mycobacteria, it is important to distinguish the genus Mycobacteria, and it is known that mycobacteria contain rpoB genes in common and can be used for identification of species (Lee Hye-young et al., Korean patent publication) No. 10-2001-0038701).

However, since the patent uses the PCR-RFLP method, it is difficult to use it for early detection of Mycobacterium tuberculosis and Mycobacteria due to problems such as cultivation due to low sensitivity or complex experimental steps. There is an inadequate disadvantage.

Therefore, the inventors of the present application intend to use an analytical method that can exert a high sensitivity to the genus Mycobacteria, such as real time PCR, and can be performed quickly and easily.

However, the primers for detecting rpoB genes known to date form very large amplification products (340-360 bp), and thus are not suitable for real time polymerase chain reaction.

Thus, the present inventors have developed primers that form amplification products of a size suitable for real-time polymerase chain reaction. That is, the primers of SEQ ID NO: 3 and primers of SEQ ID NO: 4 are primers targeting the rpoB gene site, which is a specific gene site in mycobacteria, and these primers form PCR amplification products of 100 bp or less. Therefore, the use of these primers has the advantage of being very reliable and can quickly detect the mycobacteria genus.

The composition for detecting Mycobacterium tuberculosis or Mycobacteria according to the present invention may optionally further comprise an internal control primer. This internal control is a false negative problem, that is, to determine whether the PCR reaction was properly performed, and can arbitrarily select genes that are normally expressed regardless of the presence of Mycobacterium tuberculosis or mycobacteria.

In one preferred example, the internal control primer may be a plant derived gene specific primer added in all samples.

The plant-derived gene may be preferably a lectin, and the lectin gene specific primer may be a primer including a nucleotide sequence of SEQ ID NO: 28 or a primer including a nucleotide sequence of SEQ ID NO: 29.

In the present invention, each base sequence in the primer of SEQ ID NO: 1 to the primer of SEQ ID NO: 4, the primer of SEQ ID NO: 28, and the primer of SEQ ID NO: 29 are as shown in Table 1 below.

[Table 1] Base sequence of primer

As described above, the present invention is a novel primer specific for Mycobacterium tuberculosis IS6110 gene, for detecting Mycobacterium tuberculosis comprising a sense primer comprising the nucleotide sequence of SEQ ID NO: 1 and / or an antisense primer comprising the nucleotide sequence of SEQ ID NO: 2. To provide a composition.

In addition, the present invention as a primer specific for the rpoB gene of mycobacteria, mycobacterial genus composition comprising a sense primer comprising the nucleotide sequence of SEQ ID NO: 3 and / or an antisense primer comprising the nucleotide sequence of SEQ ID NO: 4 To provide.

Primers specific for the rpoB gene of the mycobacteria form a PCR amplification product having a length of 100 bp or less suitable for real time polymerase chain reaction.

In one preferred embodiment, the mycobacteria may be mycobacterium tuberculosis or mycobacterium tuberculosis mycobacterium bovis, bar r can be further improved by detecting the rpoB gene with the IS6110 gene of the tuberculosis group.

In another preferred embodiment, the mycobacteria may be nontuberculous mycobacteria (NTM), in this case, by the sequencing of the rpoB gene can determine the presence and type of non-tuberculosis mycobacteria, not TB group There is an advantage.

Among the non-tuberculosis mycobacteria, the rpoB gene sequence is known as in FIG. 1.

In addition to the mycobacteria according to Figure 1, the inventors of the present invention, mycobacteria, mycobacterium acapulsensis (M. acapulsensis), mycobacterium flavescens (M. flavescens), mycobacterium gastin (M. gastin), Mycobacterium intracellulare (M. intracellulare), Mycobacterium Kansasii, Mycobacterium Fulveris, Mycobacterium simie (M. simiae), M. xenopi Schwabacher, Mycobacterium austroafricanum, Mycobacterium celatum, Mycobacterium gastri (M) gastri, Mycobacterium Gordonae, Mycobacterium Agri, Mycobacterium Asiaticum, Mycobacterium Sekatum ), Mycobacterium Diernhoferi, Mycobacterium M. fortuitum, Mycobacte Partial rpoB gene sequences of M. nonchromogenicum, Mycobacterium play (M. phlei), and M. genavense were identified.

In addition, the inventors of the present application confirmed that the primers of the nucleotide sequences of SEQ ID NOs: 3 and 4 are effective for identifying 42 kinds of mycobacteria and have specificity.

2. Probe for the detection of Mycobacterium tuberculosis or Mycobacteria

In another aspect, the present invention provides a probe comprising a nucleotide sequence of SEQ ID NO: 5; A probe comprising a nucleotide sequence of SEQ ID NO: 6; It provides a composition for the detection of Mycobacterium tuberculosis or Mycobacteria comprising one or two or more probes selected from the group consisting of; and a probe comprising a nucleotide sequence of SEQ ID NO: 7.

The composition may optionally further comprise an internal control probe, wherein the internal control probe preferably comprises a nucleotide sequence of SEQ ID NO: 30 as a lectin gene specific probe that is a plant-derived gene. It may be a probe.

In the present invention, the "probe including the nucleotide sequence of SEQ ID NO: x" is a concept including a nucleotide sequence each having a sequence homology of 95% or more. In the present specification, for the convenience of description, the "probe including the nucleotide sequence of SEQ ID NO x" is abbreviated as "the probe of SEQ ID NO x".

The probe of SEQ ID NO: 5 specifically reacts with the IS6110 gene region, and the probe of SEQ ID NO: 6 and the probe of SEQ ID NO: 7 are probes that commonly respond to rpoB genes in all detectable mycobacteria.

As described above, probes specific to the IS6110 gene and the rpoB gene can more clearly detect whether the mycobacteria in the sample are Mycobacterium tuberculosis, and the presence or absence of the mycobacterial bacterium can be confirmed by confirming the existence of the rpoB gene.

In particular, a probe specific for the IS6110 gene or the rpoB gene according to the present invention may be usefully used for PCR quantitative analysis by a Taqman assay or a molecular beacon assay.

Thus, in one preferred embodiment, the probe is labeled with a fluorescent material at the 5 'end and a 3' end, respectively, a fluorescent material (reporter) labeled at the 5 'end and a fluorescent material (quencher) labeled at the 3' end May indicate a mutual interference phenomenon. Accordingly, the color of the probe is restricted when the probe is bound to the IS6110 gene or the rpoB gene present in the sample, and the fluorescent substance labeled at the 5 'end is lost while the probe is degraded when the polymerase chain reaction is performed. The fluorescent material labeled on the color reaction.

The fluorescent substance labeled at the 5 'end is not particularly limited, and for example, 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX). ), Tetrachloro-6-carboxyfluorescein, and Cyanine-5 (Cy5), but is not limited thereto.

In addition, the fluorescent substance labeled at the 3 'end may be 6-carboxytetramethyl-rhodamine (AMRA) or black hole quencher-1,2,3 (BHQ-1,2,3). However, the present invention is not limited thereto.

In the present invention, each base sequence in the probe of SEQ ID NO: 5 to the probe of SEQ ID NO: 7, and the probe of SEQ ID NO: 30 are as shown in Table 2 below.

[Table 2] base sequence of the probe

3. Compositions and kits for real time PCR analysis

The present invention also provides a composition for analysis of Mycobacterium tuberculosis and / or Mycobacteria by real time PCR analysis, comprising: a primer comprising the nucleotide sequence of SEQ ID NO: 1; A primer comprising a nucleotide sequence of SEQ ID NO: 2; A primer comprising a nucleotide sequence of SEQ ID NO: 3; And a primer comprising a nucleotide sequence of SEQ ID NO: 4; one or two or more primers selected from the group consisting of: a probe comprising a nucleotide sequence of SEQ ID NO: 5; A probe comprising a nucleotide sequence of SEQ ID NO: 6; And one or two or more probes selected from the group consisting of; a probe comprising a nucleotide sequence of SEQ ID NO: 7;

The composition may also further comprise an internal control primer and an internal control probe for the purpose of preventing false negatives.

The internal control primer may be a sense primer comprising a nucleotide sequence of SEQ ID NO: 28 or an antisense primer comprising a nucleotide sequence of SEQ ID NO: 29, and the internal control probe may be a probe comprising a nucleotide sequence of SEQ ID NO: 30.

That is, the composition for quantitative analysis according to the present invention includes the above-described primer for detecting Mycobacterium tuberculosis or non-tuberculosis mycobacteria, Mycobacterium tuberculosis or non-tuberculosis mycobacteria, and optionally an internal control primer and probe.

Real time PCR for amplifying two or three genes simultaneously in one tube with DNA extracted from a clinical sample using the composition according to the present invention and analyzing the detected product in real time Analysis can be performed.

In one preferred embodiment, the composition is

primers specific for the Mycobacterium tuberculosis IS6110 gene comprising: (i) a sense primer comprising the nucleotide sequence of SEQ ID NO: 1 and an antisense primer comprising the nucleotide sequence of SEQ ID NO: 2; and a probe comprising the nucleotide sequence of SEQ ID NO: 5; and Probe mixtures;

(ii) a sense primer comprising the nucleotide sequence of SEQ ID NO: 3 and an antisense primer comprising the nucleotide sequence of SEQ ID NO: 4; and a probe comprising the nucleotide sequence of SEQ ID NO: 6 and / or a nucleotide sequence of SEQ ID NO: 7 Primer and probe mixture specific for the mycobacteria gene rpoB gene, including a probe;

(iii) primers and probes specific for an internal control gene comprising a sense primer comprising the nucleotide sequence of SEQ ID NO: 28 and an antisense primer comprising the nucleotide sequence of SEQ ID NO: 29; and a probe comprising the nucleotide sequence of SEQ ID NO: 30 mixture; And

(iv) a PCR reaction mixture comprising buffer, DNA polymerase, dNTP and sterile distilled water; It may include.

The analytical composition includes the three pairs of primers and three probes. The inventors of the present application perform real-time multiplex PCR using such a composition to detect and diagnose Mycobacterium tuberculosis and mycobacteria in the specimen, and to determine the possibility of false positives. It was confirmed that almost 100% can be excluded.

Specifically, the mixture (i) is capable of amplifying only the tuberculosis bacteria group and is specific for an IS6110 gene site amplified using the primer and a primer having a very high sensitivity of 97% or more and a 99% positive predictive value for the IS6110 gene site. And probes that bind to each other.

In addition, the mixture (ii) can amplify the rpoB gene, which is a specific gene of the mycobacteria, and amplifies the primer and the primer to form a rpoB gene region having a length suitable for analysis by real-time polymerase chain reaction. And a probe that specifically binds to the rpoB gene site.

At this time, since the probe is bound to the rpoB gene of all detectable mycobacteria, it is possible to determine the presence or absence of mycobacteria.

In some cases, one or more probes including rpoB gene regions representing different sequences according to the type of mycobacteria may be additionally included. In this case, you can check the types of mycobacteria corresponding to each.

Therefore, when using the assay kit comprising the composition, it is possible to quantify tuberculosis-specific genes more easily and accurately, and to confirm the presence of non-tuberculosis mycobacteria, which can be widely used for accurate diagnosis of tuberculosis. .

Such real time PCR analysis can be performed using a commercially available real time polymerase chain reaction device, and the real time polymerase chain reaction device is, for example, a SLAN real time PCR detection system (LG). Life Science, Korea), LightCyclerTM (Roche, Germany), ABI PRISMTM 7000/7700 (Applied Biosystems, USA), iCyclerTM (Bio-Rad, USA), Rotor-GeneTM (Corbett, Australia), OpticonTM (PharmaTech, USA), etc. It may include, but is not limited thereto.

4. Methods of analysis of genus Mycobacterium tuberculosis or Mycobacteria

The analytical composition according to the present invention may be used for qualitative analysis for identifying the presence of Mycobacterium tuberculosis or Mycobacteria, or may be used for quantitative analysis thereof. That is, the present invention provides a method for quantitative or qualitative analysis of Mycobacterium tuberculosis or non-tuberculosis mycobacteria using the analytical composition.

Qualitative analysis to identify the presence of the Mycobacterium tuberculosis bacteria or mycobacteria can be performed by performing a real-time polymerization PCR using the analytical composition to confirm the time point when the peak is observed.

In addition, the quantitative analysis may be performed by comparison with a standard curve. In one preferred example, the quantitative analysis may be performed using the analytical composition through the following steps.

(1) preparing a genetic analysis sample by mixing the DNA of the bacteria extracted from the sample to the analysis composition;

(2) performing real-time polymerization PCR on the genetic analysis sample of step (1) to obtain a PCR product;

(3) quantifying the PCR product of step (2) to obtain a quantitative curve; And

(4) quantifying the IS6110 gene, rpoB gene and internal control gene specific gene present in the DNA of the bacteria extracted from the sample using the above quantitative curve;

That is, a quantification comprising primer pairs and probes specific for the Mycobacterium tuberculosis IS6110 gene, primer pairs and probes specific for the rpoB gene of the mycobacteria genus, and three pairs of primer pairs and probes specific to the internal control gene and three probes. When the DNA of the bacteria extracted from the sample was added to the analytical composition and subjected to real-time polymerization PCR, primer pairs and probes specific to internal control genes such as IS6110 gene, rpoB gene, and lectin gene were attached to the DNA. Is amplified.

As a method for quantifying the PCR product, for example, when using the Techman assay, the IS6110 gene, the rpoB gene, and the internal parts of the IS6110 gene, the rpoB gene, and the lectin gene are decomposed by confirming whether the amplification of the gene is emitted. Control gene specific genes can be quantified.

5. RpoB Gene-Specific Nucleic Acids in Mycobacteria

The present invention also provides a rpoB gene specific nucleic acid of the genus Mycobacteria comprising one or more selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 8 to 27.

Mycobacterium acapulsensis specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 8;

M. flavescens specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9;

Mycobacterium gastin (M. gastin) specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 10;

M. intracellulare specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11;

M. kansasii specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12;

M. pulveris specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 13;

M. simiae specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 14;

M. xenopi Schwabacher specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 15;

M. austroafricanum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 16;

M. celatum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 17;

Mycobacterium gastri (M. gastri) specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 18;

M. gordonae specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 19;

A M. agri specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20;

Mycobacterium asiaticum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 21;

Mycobacterium sekatum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 22;

Mycobacterium diernhoferi specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 23;

Mycobacterium M. fortuitum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 24;

Mycobacterium nonchromogenicum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 25;

Mycobacterium play (M. phlei) specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 26; And

M. genavense specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 27;

All of these nucleic acids include the rpoB gene, and these base sequences can be used to make primers or probes that commonly react in mycobacteria, or to easily prepare primers or probes having specificity for each mycobacteria species.

Hereinafter, the present invention will be further described with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Primer and Probe

IS6110 specific primers and probes as used in the present invention, the National Institutes of Health under analysis using the program DNAsis's genebank NC000962 the nucleotide sequence hitachi software (s) in the gene bank (www.ncbi.nlm.nih .gov) operated by NCBI After determining the nucleotide sequence, it was again analyzed by BLAST ( www.ncbi.nlm.nih.gov/BLAST/ ) to confirm that it was a primer nucleotide sequence capable of amplifying only tuberculosis bacteria group.

In addition, the rpoB specific primers and probes used in the present invention, after obtaining the mycobacterial rpoB gene from the entrez of the genebank, using clustal X to analyze the conserved site (see Fig. 1), and then determine the base sequence thereof, and again BLAST ( www.ncbi.nlm.nih.gov/BLAST/ ) was confirmed to be a primer sequence that can only amplify the genus Mycobacteria.

Since the standard strain was used to secure the base sequence of another unknown species (see FIG. 2), it was confirmed that the gene of the standard strain newly obtained by determining the primer can be amplified.

Plant-derived gene specific primers and probes have obtained the nucleotide sequence from the previously published patent.

Example 2 Synthesis of the Primer

The primers analyzed in Example 1 were metabion using a method such as oligonucleotide synthesis described in 10.42 of molecular cloning 3rd edition (sambrook and rusell, cold spring harbor laboratory press, New York, USA, 2001). , Germany).

Example 3 Extraction of Bacteria DNA from Clinical Specimens or Standard Strain Cultures

1 to 2 ml of sputum or standard strain culture of patients suspected of tuberculosis and the same amount of 4N NaOH in a 15 ml tube was sufficiently stirred and centrifuged at 4,000 rpm for 20 minutes. The supernatant was removed, and 10 ml of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH 2 PO ₄ ) was added to the precipitate, followed by well stirring, followed by centrifugation at 4,000 rpm for 20 minutes. The supernatant was removed again, the precipitate was transferred to a 1.5 ml tube, 1 ml of PBS buffer was added thereto, stirred, and centrifuged at 13,000 rpm for 5 minutes. The supernatant was removed again, and 50% ul of 5% (w / v) Chelex 100 resin (Bio-Rad) was added to the precipitate, heated at 100 ° C for 20 minutes, and then centrifuged at 13,000 rpm for 3 minutes. DNA supernatant was used as template DNA for PCR.

Example 4 Real-Time Multiple PCR Reaction Analysis Using the Primer and Probe

(1) The PCR reaction composition was prepared using the PCR reaction composition as shown in Table 3, and the PCR reaction was performed in the SLAN real time PCR detection system (LG Life Science, Korea) under the conditions as shown in Table 4.

(2) The reaction product was measured in real time, and after the reaction was completed, the result was analyzed using the SLAN 7.0 program.

(3) Figure 2 can be determined to be tuberculosis bacteria positive, Figure 3 is mycobacteria genus positive, Figure 4 negative.

[Table 3] Real time PCR reaction composition

[Table 4] Real time PCR reaction conditions

[Example 6] Comparison of diagnostic effects on Mycobacterium tuberculosis and Mycobacteria genus of the culture method (MGIT, BD, USA) and the kit of the present invention

The diagnostic effect of Mycobacterium tuberculosis and Mycobacteria genus was compared using a culture method (MGIT, BD, USA) and the kit of the present invention.

The culture method used for comparison was carried out according to the manufacturer's instructions. For cultured positive samples, species were identified through amplicor MTB kit (Roche Diagnostics, USA) and sequencing. The results were shown in Table 5 below.

[Table 5] Comparative Experiment Results

(A) Result table

TB: Mycobacterium tuberculosis / NTM: Mycobacterium tuberculosis

(B) sensitivity and specificity

In the above description, 'sensitivity' means a rate of positively detecting a person with a disease, that is, a rate of judging a person with a disease as having a disease. 'Specificity' refers to a rate of negatively detecting a healthy person, that is, a rate of determining a normal person as normal. The positive predictive rate refers to the probability that a person who is positive by the diagnostic method has the disease. 'Negative predictive rate' refers to the probability that a person who is negative by a diagnostic method does not have the disease.

According to Table 5, the results of the RT-PCR using the composition according to the present invention shows almost the same reliability as the experimental results carried out by the culture method of the tuberculosis and non-tuberculosis bacterium analysis method currently showing the highest reliability You can clearly see the point. In particular, the sensitivity to Mycobacterium tuberculosis was found to be 97% or more, and the specificity was also 99%.

Example 7 Comparative Study of Diagnostic Effects of Mycobacterium Tuberculosis and Mycobacteria Genus of AFB staining and Kit of the Present Invention

For the samples obtained in Example 3, the results obtained using the AFB staining method, which was previously used for the identification of non-tuberculosis mycobacteria (NTM), and the results obtained by the RT-PCR method using the kit according to the present invention The comparison is shown in Table 6 below.

[Table 6] Comparative Experiment Results

In Table 6, (-) means NTM negative, (+) means NTM positive, the more + means that the higher the NTM concentration.

As shown in Table 6, it can be seen that the same in the RT-PCR results according to the present invention in all samples showing NTM positive using AFB staining method. In addition, in the AFB staining method, according to the RT-PCR results according to the present invention in the sample determined to be NTM negative, it can be confirmed that there are as many as three samples that appear positive.

From the above results, it can be seen that when using the composition according to the present invention, NTM can be discriminated with much higher sensitivity and reliability than AFB staining method, which is the most widely used NTM analysis method.

Example 8 Results of RT-PCR Performance of the Invention on Various Mycobacteria

A total of 42 mycobacteria were subjected to RT-PCR according to the methods of Examples 3 and 4, and the results are shown in Table 7 below.

TABLE 7

In Table 7, above, the numerical value is less than 35, indicating positive, and IC represents the internal control. In addition, Ct means the number of cycles that pass the rt-pcr result threshold as a threshold cycle value, and No Ct means that there is no threshold cycle.

As shown in Table 7, RT-PCR performance according to the present invention showed that M. tuberculosis H37Rv, M. bovis, M. bovis BCG, M. africanum, M. microti belonging to all the tuberculosis group was positive for Mycobacterium tuberculosis. The remaining mycobacteria were found to be mycobacteria positive. Therefore, it can be clearly confirmed that RT-PCR results using the assay composition according to the present invention are useful for the detection of Mycobacterium tuberculosis and Mycobacteria genus.

As described above, by performing the real-time polymerization PCR method using the analytical composition comprising the specific primer and probe according to the present invention, the detection of tuberculosis and mycobacteria genus can be performed in a shorter time with superior sensitivity and specificity. have.

1 is a result of aligning some nucleotide sequences of the rpoB gene of the known mycobacteria;

2 is a real-time multiplex PCR result photograph of a tuberculosis group positive sample using the composition for analysis according to the present invention;

3 is a real-time multiplex PCR result photograph of a mycobacterial positive sample using the analytical composition according to the present invention;

4 is a real-time multiplex PCR result photograph of a negative sample using the composition for analysis according to the present invention.

<110> LG Life Sciences Co Ltd. <120> 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 <130> LL08KR003 (P) <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer (IS2sn) <400> 1 tgggtagcag acctcaccta tg 22 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer (IS2as) <400> 2 agcgtaggcg tcggtga 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (rpo1sn) <400> 3 gayatctayc gsaagctgcg 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer (rpo1as) <400> 4 ggtcgtarcg cttctccttg a 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe (IF23T) <400> 5 acgtaggcga accctgccca ggt 23 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe (RPO1H2T g) <400> 6 ttggtcggcg gytcgcccgg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe (RPO1H2T c) <400> 7 ttggtcgggg gytcgcccgg 20 <210> 8 <211> 510 <212> DNA <213> M. acapulsensis <400> 8 ttcgggttct ccgagatcat gatgagcacg ctggagaagg acaacaccgc cggaaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgcccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggccgctaca aggtcaacaa gaagctgggc atcaccgaga acccggcgca gaccacttcg 240 accacgctga ccgaagagga cgtcgtcgcc accatcgagt acctggtgcg gctgcatcag 300 ggcgacaaga cgatgaccgt cccgggtggt gtcgaggttc ccgtcgaggt cgatgacatc 360 gaccacttcg gcaaccgtcg tctgcgcacc gtcggcgagc tgatccagaa ccagatccgg 420 gtgggcctgt cgcgcatgga gcgcgtcgtg cgtgagcgca tgaccactca ggacgtcgag 480 gcgatcacgc cgcagaccct gatcaacatc 510 <210> 9 <211> 510 <212> DNA <213> M. flavescens <400> 9 ttcgggttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgc aggcaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtg 180 ggtcggtaca aggtcaacaa gaagctgggc atcaccgaga acccggccga cacgacctcg 240 accacgctga ccgaagagga cgtcgtcgcc accatcgagt acctggtgcg gctgcatcag 300 ggcgacaaga cgatgaccgt cccgggtgga gtcgaggtgc ccgtcgaggt cgacgacatc 360 gaccacttcg gtaaccgtcg tctgcgcacc gtcggcgagc tgatccagaa ccagatccgg 420 gtcggcctgt cgcggatgga gcgcgtcgtc cgtgagcgga tgaccaccca ggacgtcgag 480 gcgatcacgc cgcagaccct gatcaacatc 510 <210> 10 <211> 507 <212> DNA <213> M. gastin_wayne <400> 10 ttcggcttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgc cggcaccgat 60 gaggcgctgc tggacatcta tcgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggccgctaca aggtcaacaa gaagctgggg ctgcacgccg gcgagccgat cacgtcgtcg 240 acgttgaccg aggaagacgc cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagccgacga tgacggttcc gggcggcgtc gaggtgccgg tggaaaccga cgacatcgac 360 cacttcggca atcggcggct gcgcaccgtg ggcgaactga tccagaacca gatccgggtc 420 ggcatgtcgc gcatggagcg tgttgtccgt gagcggatga ccactcagga cgtcgaggcc 480 atcacgccgc agaccttgat caacatc 507 <210> 11 <211> 507 <212> DNA <213> M. intracellulare <400> 11 ttcggcttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgc cggcaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcgcgtgtc 180 ggccgctaca agatcaacaa gaagctcggc ctgcacgcgg gcgagccgat caccagctcg 240 acgctgaccg aggaagacgt cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagcccacga tgaccgtccc cggcggcatc gaggtgccgg tggagaccga cgacatcgac 360 cacttcggca accgccgcct gcgcaccgtg ggtgagctga tccagaacca gatccgggtc 420 ggcatgtcgc ggatggagcg cgtcgtccgc gagcggatga ccacgcagga cgtcgaggcc 480 atcacgccgc agaccctgat caacatc 507 <210> 12 <211> 507 <212> DNA <213> M. kansasii <400> 12 ttcggcttct ccgagatcat gatgtcgacg ctggaaaagg acaacaccgc cggcaccgac 60 gaagccctgc tggacatcta ccgaaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcccagaccc tactggagaa cctgttcttc aaggagaagc gctacgacct ggcccgtgtc 180 ggccgataca aggtcaacaa gaagctgggc ctgaacacca atcatccgat caccacgacg 240 acgctgaccg aagaagacgt cgtcgccacc atcgagtatc tggtccgcct gcacgagggc 300 caggccacga tgaccgtgcc gggcggggtc gaggtgccgg tggaaaccga cggcatcgac 360 cacttcggca accgccggtt gcgtaccgtc ggcgagctga tccagaacca gatccgggtc 420 ggcatgtcga ggatggagcg ggtggtccgg gaacggatga ccactcagga cgtcgaggcg 480 atcacgccgc agacgttgat caacatc 507 <210> 13 <211> 507 <212> DNA <213> M. pulveris <400> 13 ttcgggttct ccgagatcat gatgagcacg ctggagaagg acaacaccgc gggcaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgcccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggccgctaca aggtcaacaa gaagctgggc atgcatcccg gtgagccgat cgagacgacc 240 acgctcaccg aagaggacat cgtcgccacc atcgagtacc tggtgcggct gcatcagggc 300 gacgcgacga tgacggtgcc cggcggcgcc gaggtccctg ttgaagtgga tgacatcgac 360 cacttcggca accggcgcct gcgcacggtc ggcgagctga tccagaacca gatccgggtc 420 ggcctgtcgc gcatggagcg cgtcgtgcgc gagcgcatga ccacccagga cgtggaggcg 480 atcacgccgc agaccctgat caacatc 507 <210> 14 <211> 507 <212> DNA <213> M. simiae_karassove <400> 14 ttcggcttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgc cggcaccgat 60 gaggcgctgc tggacatcta tcgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggccgctaca aggtcaacaa gaagctgggg ctgcacgccg gcgagccgat cacgtcgtcg 240 acgttgaccg aggaagacgt cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagccgacga tgacggttcc gggcggcgtc gaggtgccgg tggaaaccga cgacatcgac 360 cacttcggca atcggcggct gcgcaccgtg ggcgaactga tccagaacca gatccgggtc 420 ggcatgtcgc gcatggagcg tgttgtccgt gagcggatga ccactcagga cgtcgaggcc 480 atcacgccgc agaccctgat caacatc 507 <210> 15 <211> 507 <212> DNA <213> M. xenopi_schwabacher <400> 15 ttcggcttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgt cggcagcgac 60 gacgcgttgc tggacatcta ccgcaagctg cggccgggcg aaccgccgac caaggagtcg 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgggtg 180 ggccgctaca aggtcaacaa gaaactcggg ctgaacaccg agaatgcgcc aaccaccacg 240 accctgaccg aagaggacgt cgtcgccacc atcgaatacc tggtgcgctt gcacgagggg 300 cacgccacga tgaaggtccc cggtggcgtc gaggtgccgg tggagaccga cgacatcgac 360 cacttcggca accggcggct gcgcacggtc ggcgagctga tccaaaacca gatccgggtc 420 ggcatgtcga ggatggagcg ggtggtccgc gagcggatga ccactcagga cgtcgaggcg 480 atcaccccgc agacgttgat caacatc 507 <210> 16 <211> 507 <212> DNA <213> M. africanum <400> 16 ttcgggttct ccgagatcat gcgatcgacg ctggagaagg acaacaccgt cggcaccgac 60 gaggcgctgt tggataccta ccgcaagctg cgtccgggcg agcccccgac caaagagtca 120 gcgcagacgc tgttggaaaa cttgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggtcgctata aggtcaacaa gaagctcggg ctgcatgtcg gcgagcccat cacgtcgtcg 240 acgctgaccg aagaagacgt cgtggccacc atcgaatatc tggtccgctt gcacgagggt 300 cagaccacga tgatcgttcc gggcggcgtc gaggtgccgg tggaaaccga cgacatcgac 360 cacttcggca accgccgcct gcgtacggtc ggcgagctga tccaaaacca gatccaggtc 420 ggcatgttgc ggatggagcg ggtggtccgg gagcggatga ccacccagga cgtggaggcg 480 atcacaccgc agaccctgat caacatc 507 <210> 17 <211> 507 <212> DNA <213> M. celatum <400> 17 ttcgggttct ccgagatcat gatgagcacc ctggagaagg acaacaccgc caaccaggac 60 gaggcgctgc tcgacatcta ccgcaagctg cggccgggcg aaccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct cgcccgcgtc 180 ggccgctaca aggtcaacaa gaagctcggt ctgcaccccg gtgagccgat cgtcaccacc 240 acgctgaccg aggaggacgt cgccgccacc atcgagtacc tggtgcggct gcatcagggc 300 gacaagacca tgtgcgtgcc cggcggcgtc gaggtgccgg tcgaggtgga cgacatcgac 360 cacttcggca accgccggct gcgcaccgtc ggcgagctga tccagaacca gatccgggtc 420 ggcctgtcgc gtatggagcg cgtcgtgcgc gagcgcatga ccacccagga cgtcgaggcg 480 atcacgccgc agaccctgat caacatc 507 <210> 18 <211> 522 <212> DNA <213> M. gastri <400> 18 ttcgggttct ccgagatcat gatgtcgacg ctggaaaagg acaacaccgc cggcaccgac 60 gaagcgctgc tggacatcta ccgcaagctg cgcccgggcg agccgctgac caaggagtcg 120 gcccagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggccgctaca aggtcagcaa gaagctgggc ctgaacaccg atcatccgat caccaccacg 240 acgctgaccg aggaagacgt cgtcgccacc atcgagtacc tggttcgcct gcaccacgcc 300 tctcagggtg gccaggcccc cgttatgact gtccccggcg gggtcgaggt gccggtggaa 360 accgacgaca tcgaccactt cggcaaccgc cggctgcgca cggtgggcga gctgatccag 420 aaccagatcc gggtcggcat gtccaggatg gagcgcgtcg tccgggagca gatgaccact 480 caggacgtcg aggccatcac gccgcagacc ctgatcaaca tc 522 <210> 19 <211> 507 <212> DNA <213> M. gordonae <400> 19 ttcgggttct ccgagatcat gatggggacg ctggagaagg acaacaccgc aggcaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccgggcg aaccccccac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgggta 180 ggccgctaca aggtcaacaa gaagctcggc ctgcacgtcg gcgatccgat caccagctcc 240 acgctgaccg aggaagacgt cgtcgccacc atcgagtacc tggtccgcct gcacgagggc 300 cagcacacga tgaccgtccc gggcggcacc gaggtgccgg ttgagaccga cgacatcgac 360 cacttcggca accgccggtt gcgtaccgtg ggcgagctga tccagaacca gatccgggtc 420 ggcatgtccc gcatggagcg cgtcgtccgc gagcggatga ccactcagga cgtcgaggcg 480 atcacgccgc agacgctgat caacatc 507 <210> 20 <211> 507 <212> DNA <213> M. agri <400> 20 ttcgggttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgc aggcaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct cgcacgcgtc 180 ggccgctaca aggtcaacaa gaagctcggc ctgcacccgg gtgagcccat cggcaccacg 240 acgctgaccg aagaggacgt cgtcgccacc atcgagtacc tggtgcggct gcacgagggc 300 cagcccacga tgaccgcccc cggcggcgtc gaggttccgg tcgaggtgga cgacatcgac 360 cacttcggca accgtcgcct gcgcaccgtc ggcgagctga tccagaacca gatccgggtc 420 ggcctgtccc gcatggagcg cgtcgtgcgc gagcgcatga ccacccagga cgtcgaggcg 480 atcacgccgc agaccttgat caacatc 507 <210> 21 <211> 507 <212> DNA <213> M. asiaticum <400> 21 ttcgggttct ccgagatcat gatgggcacc ctggagaagg acagcaccgc cggtcccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtg 180 ggccgctaca aggtcaacaa gaagctgggc ctgaacgccg gccagccgat cacgtcgtcg 240 actctgaccg aggaagacgt cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagaccacga tgaccgtccc cggcggcgtc gaggtcccgg tcgaggtggt cgacatcgac 360 cacttcggta accgtcgtct gcgcaccgtg ggcgagctga tccagaacca gatccgcgtc 420 ggcctgtccc gcatggagca cgtcgtgcgt gagcgcatga ccacccagga cgtcgaggcg 480 atcaccccgc agaccctgat caacatc 507 <210> 22 <211> 507 <212> DNA <213> M. cekatum_butler <400> 22 ttcggcttct ccgagatcat gatgtcgacg ctggagaagg acaacacggt cggcaccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgcccgggcg agccgcccac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct cgcgcgggtg 180 ggccgctaca aggtcaacaa gaagctcggc ctgaacaccg cgtccccgat cacgacgacc 240 actctgaccg aagaggacgt cgtcgccacc atcgagtacc tggtccgcct gcacgagggc 300 cacaccacga tgaccgtccc gggcggagtc gaggtgccgg tggaaaccga cgacatcgac 360 cacttcggta accggcgcat tcgtaccgtc ggtgagctga tccagaacca gatccgagtc 420 ggcatgtccc gcatggagcg ggtggtccgc gagcggatga ccactcagga cgtcgaggcg 480 atcacgccgc agaccttgat caacatc 507 <210> 23 <211> 507 <212> DNA <213> M. diernhoferi <400> 23 ttcgggttct ccgagatcat gatgggcacg ctggagaagg acaacaccgc cggcaccgat 60 gaggcgctcc tggatatcta ccggaagctg cgcccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctatgacct ggctcgcgtc 180 ggccgctaca aggtcaacaa gaagctgggc ctcaacgccg gtcagccgat caccaattcg 240 acgctgaccg aagaggacgt cgtcgcgacc atcgagtacc tggtgcgtct gcacgagggc 300 cagaccacga tgaccgcccc cggcggcgtc gaggtccccg tcgaggtcga cgacatcgac 360 cacttcggta accgtcgtct gcgcaccgtc ggcgagctga tccagaacca gatccgggtc 420 ggcctgtccc gcatggaacg cgtcgtccgc gagcggatga ccacccagga cgtcgaggcg 480 atcaccccgc agaccttgat caacatc 507 <210> 24 <211> 507 <212> DNA <213> M. fortuitum <400> 24 ttcgggttct ccgagatcat gatgggcacc ctggagaagg acagcaccgc cggtcccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccaggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtg 180 ggccgctaca aggtcaacaa gaagctgggc ctgaacgccg gccagccgat cacgtcgtcg 240 actctgaccg aggaagacgt cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagaccacga tgaccgcccc cggtggcgtc gaggtgccgg tggatgtgga cgacatcgac 360 cacttcggta accgtcgcct gcgtaccgtc ggcgagctga ttcagaacca gatccgggtc 420 ggcctgtccc gtatggagcg cgtcgtgcgt gagcgcatga ccacccagga cgtcgaggcg 480 atcaccccgc agaccttgat caacatc 507 <210> 25 <211> 507 <212> DNA <213> M. nonchromogenicum <400> 25 ttcggcttct ccgagatcat gatgggcacc ctggagaagg acagcaccgc cggtcccgac 60 gaggcgctgc tggacatcta ccgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtg 180 ggccgctaca aggtcaacaa gaagctgggc ctgaacgccg gccagccgat cacgtcgtcg 240 actctgaccg aggaagacgt cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagaccacga tgaccgtccc cggcggcgtc gaggtcccgg tcgaggtgga cgacatcgac 360 cacttcggta accgtcgtct gcgcaccgtg ggcgagctga tccagaacca gatccgcgtc 420 ggcctgtccc gcatggagcg cgtcgtgcgt gagcgcatga ccacccagga cgtcgaggcg 480 atcaccccgc agaccctgat caacatc 507 <210> 26 <211> 507 <212> DNA <213> M. phlei <400> 26 ttcgggttct ccgagatcat gatgagcacg ctggagaagg acaacaccgc caaccaggac 60 gaggcgctgc tcgacatcta ccgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct cgcccgcgtc 180 ggccgctaca aggtcaacaa gaagctgggc ctgaacccgg gtcagccgat cggcaccacc 240 acgctgaccg aagaggacgt cgtcgccacc atcgagtacc tggtgcgcct gcaccagggc 300 gacaagacga tgaccgtgcc cggcggcgtc gaggtccccg tcgaggtcga cgacatcgac 360 cacttcggca accgtcgtct gcgcaccgtc ggcgagctga tccagaacca gatccgggtc 420 ggcctgtcgc gtatggagcg cgtcgtgcgc gagcgcatga ccacccagga cgtcgaggcg 480 atcacgccgc agaccttgat caacatc 507 <210> 27 <211> 507 <212> DNA <213> M. genavense <400> 27 ttcggcttct ccgagatcat gatgtcgacg ctggagaagg acaacaccgc cggcaccgat 60 gaggcgctgc tggacatcta tcgcaagctg cgtccgggcg agccgccgac caaggagtcc 120 gcgcagaccc tgctggagaa cctgttcttc aaggagaagc gctacgacct ggcccgcgtc 180 ggccgctaca aggtcaacaa gaagctgggg ctgcacgccg gcgagccgat cacgtcgtcg 240 acgttgaccg aggaagacgt cgtcgccacc atcgagtacc tggtgcgcct gcacgagggc 300 cagccgacga tgacggctcc gggcggcgtc gaggtgccgg tggaaaccga cgacatcgac 360 cacttcggca atcggcggct gcgcaccgtg ggcgaactga tccagaacca gatccgggtc 420 ggcatgtcgc gcatggagcg tgttgtccgt gagcggatga ccactcagga cgtcgaggcc 480 atcacgccgc agaccctgat caacatc 507 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (L554sn) <400> 28 gcctcttggt tgcttctttg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (L701as) <400> 29 ttccccaggt atgtcgagtc 20 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe (LC1T) <400> 30 cccgagtggg tgaggatagg gtt 23

Claims

A primer comprising a nucleotide sequence of SEQ ID NO: 1; A primer comprising a nucleotide sequence of SEQ ID NO: 2; A primer comprising a nucleotide sequence of SEQ ID NO: 3; And a primer comprising a nucleotide sequence of SEQ ID NO: 4; a composition for detecting Mycobacterium tuberculosis or Mycobacteria comprising one or two or more primers selected from the group consisting of:

The composition of claim 1, wherein the composition further comprises an internal control primer.

According to claim 2, wherein the internal control primer is a composition for the detection of Mycobacterium tuberculosis or Mycobacteria genus, characterized in that the plant-specific gene-specific primer.

The method of claim 3, wherein the plant-derived gene is lectin (lectin), the plant-derived gene-specific primer is a primer comprising a nucleotide sequence of SEQ ID NO: 28 or a primer comprising a nucleotide sequence of SEQ ID NO: 29 Or a composition for detecting the genus Mycobacteria.

As a primer specific for Mycobacterium tuberculosis IS6110 gene, Mycobacterium tuberculosis detection composition comprising a sense primer comprising the nucleotide sequence of SEQ ID NO: 1 and / or an antisense primer comprising the nucleotide sequence of SEQ ID NO: 2.

A composition for detecting the genus Mycobacteria comprising a sense primer comprising a nucleotide sequence of SEQ ID NO: 3 and / or an antisense primer comprising a nucleotide sequence of SEQ ID NO: 4 as a primer specific for the rpoB gene in the mycobacteria.

The composition of claim 6, wherein the primer specific for the rpoB gene of the mycobacteria gene forms a PCR amplification product having a length of 100 bp or less.

A probe comprising a nucleotide sequence of SEQ ID NO: 5; A probe comprising a nucleotide sequence of SEQ ID NO: 6; And a probe comprising a nucleotide sequence of SEQ ID NO: 7; a composition for detecting Mycobacterium tuberculosis or Mycobacteria comprising one or two or more probes selected from the group consisting of:

The method of claim 8, wherein the probe is labeled with a fluorescent material at the 5 'end and the 3' end, respectively, a fluorescent material (reporter) labeled at the 5 'end and a fluorescent material (quencher) labeled at the 3' end are mutually A composition for detecting Mycobacterium tuberculosis or Mycobacteria, characterized in that it exhibits an interference phenomenon.

The method of claim 11, wherein the fluorescent material labeled at the 5 'end is 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (hexachloro-6-carboxyfluorescein (HEX), tetra The chloro-6-carboxyfluorescein (tetrachloro-6-carboxyfluorescein), and Cyanine-5 (Cy5) selected from the group consisting of, the fluorescent substance labeled at the 3 'end is 6-carboxytetramethyl- rhodamine (6 -carboxytetramethyl-rhodamine, TAMRA) or black hole quencher-1,2,3 (BHQ-1,2,3) is a composition for detecting the genus Mycobacterium tuberculosis or Mycobacteria.

A composition for use in the analysis of Mycobacterium tuberculosis or Mycobacteria by real time PCR,

A primer comprising a nucleotide sequence of SEQ ID NO: 1; A primer comprising a nucleotide sequence of SEQ ID NO: 2; A primer comprising a nucleotide sequence of SEQ ID NO: 3; And a primer comprising a nucleotide sequence of SEQ ID NO: 4; one or two or more primers selected from the group consisting of: a probe comprising a nucleotide sequence of SEQ ID NO: 5; A probe comprising a nucleotide sequence of SEQ ID NO: 6; And a probe comprising a nucleotide sequence of SEQ ID NO: 7; one or more probes selected from the group consisting of.

The composition of claim 11, wherein the composition further comprises an internal control primer and an internal control probe.

The method of claim 12, wherein the internal control primer is a sense primer comprising a nucleotide sequence of SEQ ID NO: 28 or an antisense primer comprising a nucleotide sequence of SEQ ID NO: 29, wherein the internal control probe comprises a nucleotide sequence of SEQ ID NO: 30 An analysis composition, characterized in that the probe.

The composition of claim 11, wherein the composition is

(ii) a sense primer comprising the nucleotide sequence of SEQ ID NO: 3 and an antisense primer comprising the nucleotide sequence of SEQ ID NO: 4; and a probe comprising the nucleotide sequence of SEQ ID NO: 6 and / or a nucleotide sequence of SEQ ID NO: 7 Primer and probe mixture specific for rpoB gene of the genus Mycobacteria, including probe;

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

Analytical composition comprising a.

Mycobacterium tuberculosis analysis kit comprising the composition for analysis according to claim 14.

As an analysis method of Mycobacterium tuberculosis or Mycobacteria by using the composition for analysis according to claim 14,

(1) preparing a genetic analysis sample by mixing the DNA of the bacteria extracted from the sample to the analysis composition according to claim 14;

(3) quantifying the PCR product of step (2) to obtain a quantitative curve; And

Analysis method comprising a.

M. cekatum specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 22;

M. diernhoferi specific nucleic acid comprising the nucleotide sequence of SEQ ID NO: 23;

RpoB gene specific nucleic acid of at least one mycobacteria genus selected from the group consisting of: