JP2013502922A - M.M. A composition for detecting the M. TUBERCULOSIS group and the genus MYCOBACTERIA, and multiplex real-time PCR using the same composition, M. tuberculosis group. A method for simultaneously detecting the M. TUBERCULOSIS group and the genus MYCOBACTERIA - Google Patents

Abstract

  M.M. A composition for detecting the genera M. tuberculosis and mycobacteria, comprising (i) M. tuberculosis. A M. tuberculosis specific gene, primers and / or probes targeting IS6110, (ii) a mycobacteria genus specific gene, primers targeting an inter-transcriptional spacer (ITS) and Disclosed is a composition comprising / and / or a probe, and optionally, (iii) a primer and / or probe that targets a plant-derived gene as an internal control. When real-time multiplex polymerase chain reaction is performed using this composition, nontuberculous mycobacteria (mycobacteria) and mycobacteria can be detected by a single reaction, and thus clinical diagnosis is highly reliable. Can be realized quickly and easily.

Description

  The present invention relates to M.I. A composition for distinguishably detecting the M. tuberculosis group and the genus mycobacteria, and M. by real-time multiplex polymerase chain reaction (RT PCR) using the composition. And simultaneous analysis of the genera M. tuberculosis and mycobacteria. More particularly, the present invention relates to M.I. A composition for detecting the genera M. tuberculosis and mycobacteria, comprising (i) M. tuberculosis. M. tuberculosis specific gene, primer and / or probe targeting IS6110, (ii) mycobacteria genus specific gene, inter-transcriptional spacer (ITS) It relates to a composition comprising targeted primers and / or probes, and optionally (iii) primers and / or probes that target a plant-derived gene as an internal control.

  Tuberculosis is an infectious disease having the highest mortality rate in human history, and is caused by various fungi of the genus mycobacteria having a width of 0.2 to 0.5 μm and a length of 1 to 4 μm.

  The genus Mycobacteria includes a variety of mycobacteria that infect various humans and animals, causing respiratory diseases such as tuberculosis and diseases such as epilepsy, to date about 100 Bacterial species are known (Shinnick ™ et al., Mycobacterial taxonomic. Eur. J. Clin. Microbiol. Infect Dis. 1994; 13 (11): 884-901).

  Mycobacterium tuberculosis, the most important mycobacterium responsible for tuberculosis in humans, rarely appears in mycobacterium tuberculosis, mycobacterium tuberculosis There are mycobacterium bovis, and the like. Mycobacterium leprae is known as the cause of epilepsy (Shinnick, TM and Good, RC Mycobacterial taxonomic. Eur. Clin. Microbiol. Infect. Dis. 94; 13 (11): 884-901).

  Tuberculosis is a disease with a high prevalence in developing countries, with 120,000 cases of M. per year in Korea. New cases of tuberculosis (M. tuberculosis) were recognized, and the number of new tuberculosis infections reported in 2003 reached 30,687 cases (equivalent to 64 cases per 100,000 people). Korea is an OECD member country. It has been stigmatized as the highest tuberculosis death rate among 30 countries (statistical data of Korea National Statistical Office in 2004).

  In recent years, patients with acquired immune deficiency syndrome (AIDS) and other immune deficiency syndromes, infants with relatively weak immune systems, etc. have been infected with nontuberculous mycobacteria (NTM). An increasing number of patients with atypical tuberculosis have clinical symptoms similar to those associated with M. tuberculosis infection.

  Examples of NTMs include Mycobacterium avium (M. avium-intracellulare or M. avium complex), M. avium complex. M. fortuitum, M. fortuitum M. chelonae, M.C. M. gordonae, M. et al. M. szulagai, M. et al. Kansashi, M.M. Nontuberculous mycobacteria such as M. genavense (Barnes, P. F. et al., Tuberculosis in patients. 324 (23): 1644-1650).

  Many of these NTM species are M. pneumoniae. Multi-drug resistance to M. tuberculosis therapeutics and difficult to treat (Kam, KM et al., Trends in Multidrug-Resistant Mycobacterium tuberculosis in relation to Spit to Situation of Spill to Stimulation Kong, 1989-1999.Clin.Infect.Dis.2002; 34 (3): 324-329).

  Mycobacterium avium complex (MAC), which is the most common NTM, has M. It is known to be as low as 1/10 to 1/100 of M. tuberculosis, and the American Thoracic Society (ATS) is a nontuberculous mycobacteria. (American Thoracic Society, Diagnosis and treatment of disease causated by nontuberculous mycobacteria. : S1-S25).

  Therefore, nontuberculous mycobacteria are very similar in terms of symptoms to tuberculosis mycobacteria, but in terms of the drugs to be treated, tuberculous mycobacteria. And may be different. To properly treat tuberculosis mycobacteria and non-tuberculous mycobacteria, respectively, accurately distinguish between tuberculosis mycobacteria and non-tuberculous mycobacteria And early detection and diagnosis thereof are essential.

  Common tuberculosis diagnostic methods include examination of patient clinical symptoms, tuberculin skin reaction test, X-ray imaging, M.P. There is the M. tuberculosis test and the like. The easiest method, the tuberculin test, is easy to perform, but is often false negative in cases of severe tuberculosis, measles and anergy caused by immunosuppression. About 25% of the X-ray image may vary depending on the detector's ability, so diagnosis using an X-ray image accurately identifies the detector's ability to detect abnormal shadows and abnormal shadows. It depends on the ability of the detector to judge.

  M.M. Detection of tuberculosis (M. tuberculosis) is the most accurate diagnostic method for tuberculosis, and includes smear tests, culture tests, molecular diagnostic tests and the like.

  The smear test mainly uses a stain that confirms acid resistance by Ziehl-Neelsen staining. This method makes it possible to obtain results easily and quickly, but cannot distinguish between tuberculosis mycobacteria and non-tuberculous mycobacteria and has low sensitivity.

  The culture test is sensitive enough to detect bacteria even if only about 10 bacteria are present in a 1 ml sample, and allows for the isolation and accurate diagnosis of tuberculosis mycobacteria. This method requires a long period of time, for example, requires that a well-trained researcher needs to culture and observe for about 4-8 weeks and is therefore not suitable for treatment.

BACTEC (Becton Dickinson, US) is a newly developed culture method. This method involves inoculating Bacillus into a liquid culture medium containing C14 palmitate and producing ¹⁴ CO ₂ produced by metabolism of Bacillus from a growth factor calculated using a radioisotope. Estimate the amount. This method allows results to be obtained after an average of 16 days, but has the problem of requiring facilities and specialists to handle radioisotopes.

  Electrophoresis following the polymerase chain reaction (PCR) is performed by amplifying specific gene sites within 2-3 hours, resulting in M.P. This is a method capable of quickly and accurately confirming the presence of M. tuberculosis. This method allows M.P. to be more than 95% sensitive and specific from clinical specimens within one day. It enables detection of tuberculosis (M. tuberculosis) and is therefore useful (Wilson, SM et al., Progressed a simplified polymer reaction and its application to the application of it. Clin. Microbiol. 1993; 31 (4): 776-78).

  However, this method is at a high risk of carry-over contamination and requires professionals (Noordhoek, GT et al., Sensitivity and specificity of PCR for detection of mycobacteris indusulin succinescence: laboratories.J.Clin.Microbiol.1994; 32 (2): 277-284).

  On the other hand, in the case of tuberculosis diagnosis, it is considered that the PCR test is quite sensitive and specific for smear-positive specimens, and that mycobacterial smear-positive and PCR-negative specimens can be considered infected with NTM. Should.

  In the United States, where the frequency of lung disease due to NTM is high, a sample of a mycobacterial smear-positive and PCR-positive sample is tentatively diagnosed as tuberculosis, and whether or not a PCR inhibitor is present is determined. If it is negative again, it is recommended to tentatively diagnose NTM for smear-positive and PCR-negative specimens of mycobacteria. To complete the final diagnosis of NTM infection, it takes 4-8 weeks of culture (Centers for Disease Control and Prevention (CDC). Update: Nucleic acid amplification tests for tuberculosis. MMWR Mort. 2000; 49: 593-4). Although these guidelines may detect NTM infection recently, clinically it is more useful to detect NTM directly whenever possible.

  In Korea, the incidence of tuberculosis is high and the frequency of diseases caused by NTM is low. For this reason, a smear-positive subject of mycobacteria is generally considered to be infected with tuberculosis and is generally treated with an anti-tuberculosis agent. However, in recent years, in Korea, the detection rate of NTM and diseases caused by NTM has increased, and NTM may cause diseases in people with weak immune functions. NTM is difficult to diagnose, is difficult to treat due to its high frequency of drug resistance, and has a high recurrence rate (Scientific committee in Korea circulatory disease. Recycler pharmacological biotherapy. Dis. 1995; 42: 277-94.). Accordingly, there is an increasing demand for methods for diagnosing NTMs in an identifiable manner.

  A recent method for detecting NTM is the AFB staining method, which uses a restriction enzyme and a specific probe as a commonly used method for distinguishing mycobacteria species using molecular biological methods. PCR-RFLP using PCR-hybridization that has been used.

  All of these methods have low sensitivity and are problematic in that they are pre-cultured or require complex experimental steps. Not suitable for use in the rapid detection of the genera M. tuberculosis and mycobacteria.

  Accordingly, the present invention has been made to solve the above problems and other technical problems that have not yet been solved.

  One object of the present invention is to In order to accurately diagnose M. tuberculosis, M. tuberculosis It is to provide a primer and / or probe having excellent sensitivity to a gene site IS6110 specific for M. tuberculosis.

  Another object of the present invention is to confirm the presence of the genus mycobacteria (non-tuberculous mycobacteria), and therefore, it is highly effective against ITS genes that are abundant in the genus mycobacteria. It is an object of the present invention to provide a primer and / or probe having an excellent sensitivity for detecting a specific gene site.

  Another object of the present invention is that by real-time multiplex polymerase chain reaction using the present primers and / or probes, M.I. It is intended to provide a method for simultaneously detecting the tuberculosis group (M. tuberculosis complex) and nontuberculous mycobacteria (nontuberculous mycobacteria).

1. M.M. Primers for detecting M. tuberculosis or mycobacteria genus The present invention is able to detect tuberculosis mycobacteria and non-tuberculous mycobacteria A primer comprising one or more of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2; and a primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 3-11 , Relating to the composition.

  Preferably, the present invention is a composition for distinguishably detecting tuberculosis mycobacteria and nontuberculous mycobacteria, comprising a base sequence of SEQ ID NO: 1. And a primer comprising the nucleotide sequence of SEQ ID NO: 2; or a primer comprising one or more of the nucleotide sequences of SEQ ID NO: 3-9, and one or more of the nucleotide sequences of SEQ ID NO: 10-11 Relates to a composition comprising both primers comprising

  More preferably, the present invention provides a composition for distinguishably detecting tuberculosis mycobacteria and nontuberculous mycobacteria, comprising a base sequence of SEQ ID NO: 1. A primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 3-9; and a primer composition comprising one or more of the nucleotide sequences of SEQ ID NOs: 10-11.

  As used herein, the term “primer comprising a base sequence of SEQ ID NO: x” includes a base sequence having a sequence homology of 95% or more. For example, if the primer is 20 bp, if two or more of 20 bp are different, the melting temperature will be reduced by 5 degrees or more, thus causing a bad result, whereas if only one of 20 bp is different, it is slightly lower in PCR. The same result can be obtained even if the test is performed at the annealing temperature.

  Hereinafter, in the present specification, for ease of explanation, the “primer including the base sequence of SEQ ID NO: x” is simply referred to as “primer of SEQ ID NO: x”.

  In the present specification, the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 are M.I. It is a primer that targets a gene site IS6110 gene site specific to the M. tuberculosis complex.

  In general, “M. tuberculosis” means, in a narrow sense, mycobacterium tuberculosis. In the present invention, “M. tuberculosis” broadly refers not only to Mycobacterium tuberculosis, but also to Mycobacterium bovis, Mycobacterium microti (Mycobacterium microti), and mycobacterium africanum. In some cases, M.M. M. tuberculosis refers to “M. tuberculosis group” or “TB complex”.

  The IS6110 gene is derived from M. cerevisiae such as mycobacterium tuberculosis and mycobacteria bovis. It is an insertion sequence found in the M. tuberculosis group. M.M. The M. tuberculosis group is known to contain 10-12 copies of IS6110 commonly used for tuberculosis diagnosis by PCR (Thierry D. et al., J. Clin. Microbiol. 1990; 28). (12): 2668-2673). However, Kent et al. Reported that IS6110 has a different risk of false positive depending on the location of the primer (J. Clin. Microbiol. 1995; 33 (9): 2290-2293).

  For this reason, the inventors of the present invention studied a site with a low risk of false positive by analyzing the base sequence. As a result, as can be seen from the following examples, the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 were A primer base sequence that amplifies only the IS6110 gene of the M. tuberculosis group (M. tuberculosis complex). It shows a fairly high sensitivity of 97% or more and a positive predictive value of 99% or more in the IS6110 gene site of the M. tuberculosis group. This means that the risk of false positives is quite low. No primer showing such high sensitivity and positive predictive value has been found so far.

  Therefore, when the primer of SEQ ID NO: 1, the primer of SEQ ID NO: 2, or a combination thereof is used, M.P. The tuberculosis group (M. tuberculosis complex) can be detected with high reliability.

  In order to confirm the presence / absence of non-tuberculous mycobacteria, the identification of mycobacteria is important. In this regard, the rpoB gene is common in the genus Mycobacterium, and therefore the species of mycobacteria can be identified using the presence or absence of rpoB (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 low sensitivity and the need for prior culture for complex experimental steps. For this reason, this patent is an early It is not suitable for use in the rapid detection of the M. tuberculosis complex and Mycobacteria genus.

  In order to solve these problems, the present inventors have developed a novel primer specific to the rpoB gene site that forms a PCR amplification product of 100 bp or less suitable for real-time PCR (Kang Jin Seok et al. Korea Patent Application Publication No. 2008-0090156).

  However, rpoB is a gene encoding the RNA polymerase β subunit. From the results of the specificity test of the present application, although many species of microorganisms do not exhibit positivity, symptoms of pneumonia similar to tuberculosis Nocardia microorganisms exhibiting a phenotype and rhodococcus microorganisms similar to the mycobacteria genus are positive because there are similar base sequences exhibiting a common action in several microorganisms It can be understood.

  On the other hand, in order to confirm the presence of nontuberculous mycobacteria, it is important to identify the genus mycobacteria. The genus mycobacteria includes the ITS gene and the rpoB gene, and it is therefore known to use these to identify bacterial species (Kim In Soo et al., Korean Patent No. 0725579). Specification).

  However, this patent uses a primer that forms a PCR product with a size of about 250-450 bp for PCR-reverse hybridization and is therefore not suitable for real-time PCR for rapid detection. .

  Therefore, the present inventors have developed a primer that forms an amplification product of a size suitable for real-time PCR of the ITS site. That is, the primers of SEQ ID NOs: 3 to 11 are primers targeting a gene site specific to the genus mycobacteria and an ITS gene site. These primers form a PCR amplification product having a size of 100 bp or less. For this reason, since these primers do not react with Nocardia and rhodococcus microorganisms, these primers advantageously advantageously detect the mycobacteria genus fairly reliably and quickly. It is possible to show higher specificity than before.

  In accordance with the present invention, M.I. The composition for detecting the genus M. tuberculosis or mycobacteria may optionally further comprise an internal control primer. This internal control is used to check whether a false negative problem occurs, that is, whether the PCR reaction is performed normally. M. in the sample. Regardless of the presence or absence of M. tuberculosis or mycobacteria, normally expressed genes can be randomly selected.

  In a preferred embodiment, the internal control primer may be a plant-derived gene specific primer that is added to the entire sample.

  The plant-derived gene is preferably a lectin, and the lectin gene-specific primer may be a primer containing the nucleotide sequence of SEQ ID NO: 15 or a primer containing the nucleotide sequence of SEQ ID NO: 16.

  Table 1 below shows the base sequences of the primers of SEQ ID NO: 1 to 11, the primer of SEQ ID NO: 15, and the primer of SEQ ID NO: 16 of the present invention.

  In the above sequences, “R” and “Y” are combined base sequences, which indicate “R = A + G” and “Y = C + T”, respectively.

  As described above, the present invention provides an M.I. A composition for detecting M. tuberculosis, comprising: As a novel primer specific for the IS6110 gene of M. tuberculosis, one or more of a sense primer containing the nucleotide sequence of SEQ ID NO: 1 and an antisense primer containing the nucleotide sequence of SEQ ID NO: 2 A composition comprising is provided.

  Furthermore, the present invention relates to a composition for detecting the genus mycobacteria, which is a sense primer comprising a nucleotide sequence of SEQ ID NOs: 3 to 9 as a primer specific for the ITS gene of mycobacteria. Provided is a composition comprising one or more; one or more antisense primers comprising the nucleotide sequences of SEQ ID NOs: 10-11; or a combination thereof.

  A primer specific for the ITS gene of the genus mycobacteria forms a PCR amplification product having a length of 100 bp or less suitable for real-time PCR.

  In a preferred embodiment, the mycobacteria may be mycobacterium tuberculosis or mycobacterium bovis. M.M. The reliability of detection of M. tuberculosis is confirmed by the M. tuberculosis using the ITS gene. Further improvements can be made by analyzing the IS6110 gene of the M. tuberculosis group.

  In a preferred embodiment, the mycobacteria may be non-tuberculous mycobacteria (NTM). In this case, M. can be advantageously confirmed by analysis of the base sequence of the ITS gene. It is the presence and type of nontuberculous mycobacteria rather than the M. tuberculosis group.

  Furthermore, the present inventors have confirmed that the primer containing the nucleotide sequences of SEQ ID NOs: 3 to 11 is effective for identifying the following 42 species of mycobacteria and has high specificity.

2. M.M. Probes for detecting the genus M. tuberculosis or mycobacteria In another aspect, the present invention relates to M. tuberculosis or Mycobacterium genus. A composition for detecting a genus M. tuberculosis or mycobacteria, comprising a probe comprising the nucleotide sequence of SEQ ID NO: 12; a probe comprising the nucleotide sequence of SEQ ID NO: 13; and SEQ ID NO: 14 A composition comprising one or more selected from the group consisting of probes comprising the nucleotide sequences of:

  The composition may optionally further comprise an internal control probe, which is preferably a probe specific for a plant-derived gene lectin and comprising the base sequence of SEQ ID NO: 17.

  In the present invention, the term “probe containing the nucleotide sequence of SEQ ID NO: x” includes a nucleotide sequence having a sequence homology of 95% or more. For ease of explanation, hereinafter, the “probe including the base sequence of SEQ ID NO. X” is simply referred to as “probe of SEQ ID NO. X”.

  The probe of SEQ ID NO: 12 specifically reacts with the IS6110 gene site, and the probe of SEQ ID NO: 13 and the probe of SEQ ID NO: 14 are usually probes that react with all detectable ITS genes of the genus mycobacteria. .

  For this reason, the mycobacteria in the sample is expressed by M. cerevisiae using probes specific for the IS6110 gene and the ITS gene. It is possible to more accurately detect whether or not M. tuberculosis, and by confirming the presence of the ITS gene, the presence of mycobacteria can be confirmed.

  In particular, probes specific for IS6110 gene or ITS gene according to the present invention are useful for quantitative PCR assay using taqman assay or molecular beacon assay.

  Thus, in a preferred embodiment, a fluorescent material is used to label a probe at its 5 ′ and 3 ′ ends, a fluorescent material labeled at the 5 ′ end (reporter), and a fluorescent material labeled at the 3 ′ end (quenched). Char) may interfere with each other. Therefore, when the probe binds to IS6110 or ITS gene present in the sample, the color development of the probe is suppressed, but when the polymerase chain reaction is performed, the probe is decomposed, and the fluorescent material labeled at its 5 ′ end is While quenched, the fluorescent material labeled at the 3 ′ end develops color.

  The fluorescent material labeled at the 5 ′ end of the probe is not particularly limited. For example, fluorescent materials include 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6-carboxyfluorescein. And may be selected from the group consisting of cyanine-5 (Cy5), but is not limited thereto.

  Further, 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). It is not limited.

  Table 2 below shows the base sequences of the probes of SEQ ID NOS: 12 to 14 and the probe of SEQ ID NO: 17 of the present invention.

3. Compositions and Kits for Real-Time PCR Assays The present invention uses M.p. A composition for identifying, detecting and analyzing the genus M. tuberculosis and mycobacteria, comprising one or two of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2 Provided is a composition comprising a primer comprising the above; a primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 3-11; and a probe comprising one or more of the nucleotide sequences of SEQ ID NOs: 12-14 To do.

  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 including the base sequence of SEQ ID NO: 15 or a base sequence antisense primer of SEQ ID NO: 16, and the internal control probe may be a probe including the base sequence of SEQ ID NO: 17.

  That is, the composition for quantitative analysis according to the present invention comprises Primer for detecting M. tuberculosis or nontuberculous mycobacteria, or a probe for detecting nontuberculous mycobacteria, and optionally an internal control primer and Includes probes.

  Performing a real-time PCR assay using the composition according to the present invention to amplify 2 or 3 bacterial species genes with DNA extracted from clinical specimens in a single tube and analyze and detect the resulting products in real time Can do.

In a preferred embodiment, the composition comprises
(I) a sense primer comprising the base sequence of SEQ ID NO: 1 and an antisense primer comprising the base sequence of SEQ ID NO: 2; and a probe comprising the base sequence of SEQ ID NO: 12, A mixture of primers and probes specific for the IS6110 gene of M. tuberculosis;
(Ii) a sense primer comprising the nucleotide sequence of SEQ ID NO: 3-9 and an antisense primer comprising the nucleotide sequence of SEQ ID NO: 10-11; and a probe comprising the nucleotide sequence of SEQ ID NO: 13 and / or the nucleotide sequence of SEQ ID NO: 14. A mixture of primers and probes specific for the ITS gene of the genus mycobacteria, including a probe comprising;
(Iii) a primer and probe specific for an internal control gene, comprising a sense primer comprising the base sequence of SEQ ID NO: 15 and an antisense primer comprising the base sequence of SEQ ID NO: 16; and a probe comprising the base sequence of SEQ ID NO: 17 And (iv) a PCR reaction mixture comprising buffer, DNA polymerase, dNTPs and sterile distilled water.

  The analysis composition includes a plurality of groups of primers and three groups of probes. The inventors of the present invention performed real-time multiplex PCR using this composition. As a result, the inventors have almost completely eliminated the risk of false positives and found M. It was confirmed that M. tuberculosis and mycobacteria can be detected and diagnosed.

  Specifically, the mixture (i) is M.I. A primer capable of amplifying only the M. tuberculosis group and exhibiting a fairly high sensitivity of 97% or more to the IS6110 gene site and a positive predictive value of 99% or more, and this primer And a probe that specifically binds to the IS6110 gene site amplified using

  The mixture (ii) includes a primer capable of amplifying a mycobacteria genus-specific gene, the ITS gene, and forming an ITS gene site having a length suitable for analysis using real-time PCR, and this primer And a probe that specifically binds to the ITS gene site amplified using

  This probe binds to all detectable ITS genes of the genus Mycobacteria, and thus the binding can confirm the presence of mycobacteria.

  If desired, the composition may further comprise one or more probes comprising an ITS gene site with another base sequence extracted from different strains of mycobacteria. In this case, the mycobacteria species can be confirmed.

  Therefore, if an analysis kit containing this composition is used, a tuberculosis-specific gene can be quantitatively analyzed easily and accurately, and the presence of non-tuberculous mycobacteria can be confirmed. Is possible. Therefore, this analysis kit can be widely used for accurate diagnosis of tuberculosis. In some cases, each mixture may be analyzed separately and judged as a set.

  Such a real-time PCR assay can be performed using a commercially available real-time PCR apparatus. Examples of the real-time PCR apparatus include a LAN real-time PCR detection system (LG Life Sciences, Korea), LightCycler (trademark) (Roche, Germany). ), ABI PRISM ™ 7000/7700 (Applied Biosystems, USA), iCycler ™ (Bio-Rad, USA), Rotor-Gene ™ (Corbett, Australia), Opticon ™ (PharmaTech, USA). However, it is not limited to this.

4). M.M. Methods for analysis of M. tuberculosis or mycobacteria genus The composition according to the invention for analysis comprises M. tuberculosis or mycobacteria genus. It may be used for qualitative analysis to confirm the presence of M. tuberculosis or mycobacteria genus or for quantitative analysis thereof. That is, the present invention uses M.A. Provided is a method for quantitative or qualitative analysis of tuberculosis (M. tuberculosis) or nontuberculous mycobacteria.

  M.M. Qualitative analysis for confirming the presence of the genus M. tuberculosis or mycobacteria is performed by performing real-time PCR using the composition for analysis and confirming that the peak is observed. May be.

Furthermore, the quantitative analysis may be performed by comparison with a standard curve. In a preferred embodiment, the quantitative analysis may be performed using the analytical composition by the following steps:
(1) A step of preparing a sample for gene analysis by mixing bacterial DNA extracted from the sample and the composition for analysis;
(2) A step of subjecting a sample for gene analysis to real-time PCR to obtain a PCR product;
(3) a step of quantifying a PCR product to obtain a quantitative curve; and (4) an IS6110-specific gene, an ITS-specific gene and an internal control-specific gene present in bacterial DNA extracted from a sample, Using and quantifying.

  That is, multiple groups of primers and three groups of probes, Primer pair and probe specific for the M. tuberculosis gene IS6110, primer pair and probe specific for the ITS gene of Mycobacterium genus, and specific for internal control genes such as the lectin gene When bacterial DNA extracted from a sample is added to a composition for quantitative analysis including a primer pair and a probe, and real-time PCR is performed, it is specific to IS6110 gene, ITS gene, and internal control gene such as lectin gene. Each primer pair and probe binds to DNA and a PCR product is amplified.

  When taqman analysis is used as a PCR product analysis method, the IS6110 gene, ITS gene, and lectin gene are decomposed and the amplification of the gene is confirmed using the phenomenon of luminescence to confirm IS6110-specific gene, ITS Specific genes and internal control specific genes can be analyzed.

  The above and other objects, features and other advantages of the present invention will be more clearly understood when the following detailed description is considered in conjunction with the accompanying drawings.

M. using the composition for analysis according to the present invention. It is an image which shows the result of real-time multiplex PCR of a tuberculosis (M. tuberculosis) group positive sample. It is an image which shows the result of real-time multiplex PCR of the mycobacteria (mycobacterium) positive sample using the composition for analysis by the present invention. It is an image which shows the result of the real-time multiplex PCR of a negative sample using the composition for analysis by this invention.

  The invention will now be described in more detail with reference to the following examples. These examples should not be construed to limit the scope and spirit of the invention.

Example 1: Preparation of primer and probe The nucleotide sequence was determined using DNAsis manufactured by Hitachi Software Engineering Co., Ltd., and the nucleotide sequence was analyzed using BLAST (www.ncbi.nlm.nih.gov/BLAST/). By analyzing the base sequence deposited under the accession number NC000962 in GenBank (www.ncbi.nlm.nih.gov) managed by the related organization NCBI of the National Institutes of Health (NIH), the IS6110 specific for use in the present invention Primers and probes are described in M.M. It was confirmed that the primer base sequence was capable of amplifying only the M. tuberculosis group.

  Further, a mycobacteria ITS gene is obtained from Genebank Entrez, a conserved site is analyzed using clustal X, a base sequence is determined, and BLAST (www.ncbi.nlm.nih.gov/BLAST/) is used. By analyzing the nucleotide sequence, it was confirmed that the ITS-specific primers and probes used in the present invention are also primer nucleotide sequences that can amplify only the genus Mycobacteria.

  The base sequences of plant-specific gene-specific primers and probes are obtained from patents published in the art.

Example 2: Primer Synthesis Primers analyzed in Example 1 were prepared using Molecular Cloning 3rd ed. Synthesized by Metabion Corporation (Germany) using the “oligonucleotide synthesis” method described in paragraph 10.42 of (Sambrook and Rusell, Cold Spring Harbor Laboratory Press, New York, USA, 2001).

Example 3: Extraction of Mycobacterial DNA from Clinical Specimens or Standard Culture Medium 1-2 ml of patient sputum or standard culture medium suspected of tuberculosis and 1-2 ml of 4N NaOH are placed in 15 ml tubes And centrifuged at 4,000 rpm for 20 minutes. The supernatant is 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 ) is added to the resulting precipitate, followed by thorough stirring And centrifuged at 4,000 rpm for 20 minutes. The supernatant was discarded and the precipitate was transferred to a 1.5 ml tube to which 1 ml PBS buffer was added followed by stirring and centrifuging at 13,000 rpm for 5 minutes. The supernatant was again discarded, and 50-100 ui of 5% (w / v) Chelex 100 resin (Bio-Rad Corp.) was added to the resulting precipitate, and the mixture was heated at 100 ° C. for 20 minutes and 13,000 rpm. For 3 minutes, and the separated DNA supernatant was used as PCR template DNA.

Example 4: Analysis of real-time multiplex PCR reaction using primers and probes A PCR reaction composition was prepared according to the prescription of the PCR reaction composition shown in Table 3 below, and under the reaction conditions shown in Table 4 below, the real-time of the SIP real-time PCR reaction was performed using a PCR detection system (LG Life Sciences Ltd., Korea).

  The reaction product was measured in real time, and after completion of the reaction, the results were analyzed using the SLAN 7.0 program.

  It was determined as follows: FIG. M. tuberculosis group positive, Figure 2: Mycobacteria genus positive, and Figure 3: Negative.

Example 6: M. using a conventional culture method (MGIT, BD Corp., USA) and the kit of the present invention. Comparative test of diagnostic effect of M. tuberculosis and Mycobacteria genus M. tuberculosis (MGIT, BD Corp., US) and the kit of the present invention between The diagnostic effects of M. tuberculosis and mycobacteria genus were compared.

  The culture method used for comparison was performed according to the manufacturer's instructions, and the bacterial species of the cultured positive specimens were confirmed using an amplicor MTB kit (Roche-diagnosis, USA) and nucleotide sequence analysis. The results are shown in Table 5.

  As used herein, the term “sensitivity” refers to the proportion of disease patients who have a positive test result, ie, the proportion at which a person suffering from the disease is determined to have the disease. As used herein, the term “specificity” refers to the rate at which healthy people are detected as negative, ie the rate at which normal people are determined to be normal. The term “positive predictive value” refers to the probability that a person considered positive by a diagnostic method has a corresponding disease. The term “negative predictive value” refers to the probability that a person who is positive by a diagnostic method is not the corresponding disease.

  According to the results of Table 5 above, when RT-PCR is performed using the composition according to the present invention, tuberculosis mycobacteria and non-tuberculous mycobacteria are currently most reliable. It was clearly confirmed that substantially equivalent reliability was obtained compared to high assay culture methods. In particular, M.M. The sensitivity to M. tuberculosis is 97% or more and the specificity is 99%. It is suggested that M. tuberculosis can be accurately identified.

Example 7: Comparative test of diagnostic effect between Nocardia genus microorganisms exhibiting pneumonia similar to tuberculosis and Rhodococcus microorganisms similar to mycobacteria Nocardia exhibiting symptoms similar to tuberculosis ( The results obtained using the kit according to Korean Patent Application No. 2008-0090156 for the microorganisms of the genus nocardia and rhodococcus were obtained by RT-PCR using the kit according to the present invention. Compared with the results. The results are shown in Table 6.

  As can be seen from Table 6, the present invention completely eliminates the false positive probability found in conventional patented compositions.

  It can be seen from the above results that by using the composition of the present invention, NTM can be detected with considerably higher specificity compared to prior art patents.

Example 8: Results of RT-PCR of the present invention using various mycobacteria According to the methods of Examples 3 and 4, 42 species of mycobacteria were subjected to RT-PCR. The results are shown in Table 7 below.

  (In Table 7, if the value of Mycobacterium is less than 35, Mycobacterium is defined as “positive” and IC means internal control. Furthermore, Ct is the threshold cycle, which is defined as RT -As a result of PCR, the number of cycles exceeding a critical value is meant, and No Ct means no threshold cycle.)

  As can be seen from Table 7, RT. M. tuberculosis (M. tuberculosis) group. Tuberculosis H37Rv (M. tuberculosis H37Rv), M. tuberculosis H37Rv. M. bovis, M.M. Bovis BCG (M. bovis BCG), M. M. africanum, and M. africanum All M. microti tests are M. tuberculosis was positive and the remaining mycobacteria tests were positive for mycobacteria. Therefore, RT-PCR using the analysis composition according to the present invention is performed by M.M. It is useful for the detection of the genera M. tuberculosis and mycobacteria.

  As is clear from the above, when real-time PCR is performed using the analysis composition according to the present invention, M.P. Detection of the genus M. tuberculosis and mycobacteria can be achieved with high sensitivity and specificity in a short period of time compared to conventional methods.

  While preferred embodiments of the present invention have been disclosed for purposes of illustration, those skilled in the art will recognize that various modifications, additions and modifications may be made without departing from the scope and spirit of the invention as disclosed in the appended claims. It will be understood that substitutions are possible.

Claims (20)

A composition for distinguishably detecting tuberculosis mycobacteria and nontuberculous mycobacteria, comprising:
A composition comprising a primer comprising one or more of the base sequence of SEQ ID NO: 1 and SEQ ID NO: 2; and a primer comprising one or more of the base sequences of SEQ ID NOs: 3-11. The composition comprises
The composition according to claim 1, comprising both a primer comprising the base sequence of SEQ ID NO: 1; and a primer comprising the base sequence of SEQ ID NO: 2. The composition comprises
2. A primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 3 to 9; and a primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 10 to 11. A composition according to 1. The composition comprises
A primer comprising the nucleotide sequence of SEQ ID NO: 1;
A primer comprising the base sequence of SEQ ID NO: 2;
2. A primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 3-9; and a primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 10-11. Composition.   The composition according to claim 1, further comprising an internal control primer.   The composition according to claim 5, wherein the internal control primer is a plant-specific gene-specific primer. The plant-derived gene is a lectin,
The composition according to claim 6, wherein the plant-derived gene-specific primer includes one or more of a primer including the base sequence of SEQ ID NO: 15 and a primer including the base sequence of SEQ ID NO: 16. object. M.M. As a primer specific for the IS6110 gene of M. tuberculosis, one or more of a sense primer containing the nucleotide sequence of SEQ ID NO: 1 and an antisense primer containing the nucleotide sequence of SEQ ID NO: 2 Including M. A composition for detecting M. tuberculosis. One or more of the sense primers comprising the nucleotide sequences of SEQ ID NOs: 3 to 9 as primers specific to the ITS gene of the genus mycobacteria;
A composition for detecting the genus Mycobacteria comprising one or more antisense primers comprising the nucleotide sequences of SEQ ID NOs: 10 to 11; or a combination thereof.   The composition according to claim 9, wherein the primer specific for an ITS gene belonging to the genus mycobacteria forms a PCR amplification product having a length of 100 bp or less. A probe comprising the base sequence of SEQ ID NO: 12;
A probe comprising the base sequence of SEQ ID NO: 13; and M. comprising one or more selected from the group consisting of probes comprising the base sequence of SEQ ID NO: 14. A composition for detecting the genus M. tuberculosis or mycobacteria. The probe is labeled with a fluorescent material at its 5 ′ end and 3 ′ end,
The composition according to claim 11, wherein the fluorescent material (reporter) labeled at the 5 'end and the fluorescent material (quencher) labeled at the 3' end interfere with each other. The fluorescent material labeled at the 5 ′ end is selected from the group consisting of 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6-carboxyfluorescein and cyanine-5 (Cy5). ,
The fluorescent material labeled at the 3 'end is 6-carboxytetramethyl-rhodamine (TAMRA) or black hole quencher-1,2,3 (BHQ-1,2,3). Item 13. The composition according to Item 12. Using a real-time PCR assay, M.P. A composition for identifying, detecting and analyzing the genera M. tuberculosis and mycobacteria,
A primer comprising one or more of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2;
A composition comprising a primer comprising one or more of the nucleotide sequences of SEQ ID NOs: 3 to 11; and a probe comprising one or more of the nucleotide sequences of SEQ ID NOs: 12 to 14. 15. The composition of claim 14, further comprising an internal control primer; and an internal control probe. The internal control primer is a sense primer comprising the nucleotide sequence of SEQ ID NO: 15 or an antisense primer comprising the nucleotide sequence of SEQ ID NO: 16,
The composition according to claim 15, wherein the internal control probe is a probe comprising the nucleotide sequence of SEQ ID NO: 17. The composition comprises
(I) M.M. A mixture of primers and probes specific for the IS6110 gene of M. tuberculosis,
A mixture comprising a sense primer comprising the base sequence of SEQ ID NO: 1 and an antisense primer comprising the base sequence of SEQ ID NO: 2; and a probe comprising the base sequence of SEQ ID NO: 12;
(Ii) a mixture of primers and probes specific for the ITS gene of the genus mycobacteria,
A sense primer comprising the nucleotide sequence of SEQ ID NO: 3-9 and an antisense primer comprising the nucleotide sequence of SEQ ID NO: 10-11; and a probe comprising the nucleotide sequence of SEQ ID NO: 13 and / or a probe comprising the nucleotide sequence of SEQ ID NO: 14. A mixture comprising;
(Iii) a mixture of primers and probes specific for the internal control gene,
A sense primer comprising the base sequence of SEQ ID NO: 15 and an antisense primer comprising the base sequence of SEQ ID NO: 16; and a mixture comprising a probe comprising the base sequence of SEQ ID NO: 17; and (iv) a buffer, DNA polymerase, dNTP and sterilization 15. The composition of claim 14, comprising a PCR reaction mixture comprising distilled water.   A composition comprising the analytical composition according to claim 17. M. tuberculosis analysis kit. Using the composition of claim 17, M. A method for analyzing the genus M. tuberculosis or mycobacteria, comprising:
(1) A step of preparing a sample for gene analysis by mixing bacterial DNA extracted from the sample and the composition for analysis;
(2) A step of 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) an IS6110-specific gene, an ITS-specific gene and an internal control-specific gene present in the bacterial DNA extracted from the sample, A method comprising quantifying using the quantitative curve.   An ITS gene-specific primer comprising one of the nucleotide sequences of SEQ ID NOs: 3 to 11.

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