KR101802417B1 - A multiplex PCR primer set, a composition and a kit comprising the same for diagnosing Mycobacterium tuberculosis and non-tuberculous mycobacteria - Google Patents

Abstract

The present invention relates to: a multiplex PCR primer set which can specifically amplify genes capable of simultaneously diagnosing mycobacterium tuberculosis (MTBC) and non-tuberculous mycobacteria (NTM) and genes present in the mycobacterium tuberculosis of a Beijing family; a composition and a kit for simultaneous detection of the MTBC and NTM comprising the primer set; and a method for simultaneously diagnosing MTBC and NTM comprising the primer set. By using the primer set for simultaneously diagnosing MTBC and NTM of the present invention, it is possible to effectively discern the Beijing family which is a worldwide prevalent MTBC, and at the same time, to discern the MTBC and NTM having large clinical importance and to identify species.

Description

A multiplex PCR primer set for simultaneous diagnosis of Mycobacterium tuberculosis and non-Mycobacterium tuberculosis, a composition and kit comprising the primer set, and a kit comprising the same as a kit for diagnosing Mycobacterium tuberculosis and non-tuberculous mycobacteria.

The present invention relates to a multiplex PCR primer set capable of specifically amplifying a gene specifically present in the Mycobacterium tuberculosis of the Beijing family and a gene capable of simultaneously diagnosing the tubercle bacillus and the non-mycobacteria, and a primer set A kit for simultaneous detection of Mycobacterium tuberculosis and non-mycobacteria, and a method for simultaneous detection of Mycobacterium tuberculosis and non-mycobacteria including the primer set.

More than one third of the world's population is infected with Mycobacterium tuberculosis (MTB), which is a pathogenic bacterium that causes tuberculosis (TB). The World Health Organization (WHO) recognizes tuberculosis as one of the three major infectious diseases that pose a threat to human health, with 9.6 million new tuberculosis cases a year, of which about 1.5 million people Have been reported to have died. Recently, it has become increasingly difficult to treat tuberculosis due to the increase of multi-drug-resistant tuberculosis (MDR) and extensively-drug-resistant tuberculosis (XDR), and tuberculosis (TDR; Totally-drug-resistant, the situation is getting worse. This drug-resistant tuberculosis not only leads to an increase in the cost of treatment but also lowers the treatment efficiency and threatens to develop into refractory tuberculosis.

The Beijing family with the Beijing genotype is a predominant tubercular bacillus in Asia and the former Soviet Union, mainly in China, and shares a distinct genotype. It has been confirmed that it is widely distributed in about 50% in East Asia, 70% in Korea, 20% in countries with low tuberculosis incidence such as the United States and Canada, and 13% globally. There are reports that the Beijing family is highly associated with multidrug-resistant tuberculosis and broad-based tuberculosis due to its high recurrence rate, treatment failure, and favorable tolerance acquisition capability.

It is also reported that the Beijing family is highly pathogenic and induces a low level of cytokine to avoid the Th1 type of immune response and has a higher mortality rate than the other strains when infected with the Beijing family in animal experiments. It is also known that BCG (Bacillus Calmette-Guein) vaccine is one of the few strains that has a small effect and is frequently identified not only in adult patients but also in children and adolescents with BCG vaccination. Therefore, if the rate of identification of the Beijing family among the Mycobacterium tuberculosis is high, it may become a criterion that the risk of tuberculosis can be increased.

In addition, since 1980, the incidence of non-tuberculous mycobacteria (NTM) has been increasing, and the distribution of causative agents of NTM lung disease varies from country to country. In Korea, the most common cause of pulmonary disease in Korea is Mycobacterium avium complex (MAC), which accounts for 50-60%, and M. abscessus (MAB), a rapidly growing organism, accounts for 20-30% . In the United States and Japan, the most common pathogens are MAC, 60-80%, M. kansasii , 15%, and MAB, less than 5%. Most of the NTMs are widely distributed in natural environments such as natural waters and soils and have low virulence, so there is no risk of infection among humans. However, since there is no proper remedy and NTM is resistant to each antibiotic resistance, This is important. Thus, in order to efficiently treat pulmonary diseases caused by mycobacteria, a technique for accurately diagnosing at least certain species of bacteria is required.

The most commonly used method for diagnosing mycobacteria is pulmonary tuberculosis, which has been used to identify tuberculosis in the past. However, due to the recent increase in pulmonary disease caused by non - tuberculous mycobacteria, It is urgently required to develop a diagnostic technique that accurately identifies the major non-tuberculous mycobacteria causing pulmonary disease and discriminates the Beijing family of highly pathogenic M. tuberculosis. Although many diagnostic techniques have been developed in recent years, the development of molecular biologic diagnostic techniques that accurately reflect these clinical needs is still lacking.

Thus, the present inventors have conducted a multiplex PCR that includes genetic markers specifically present in the Mycobacterium tuberculosis of the Beijing family, which includes gene markers specifically present in Mycobacterium tuberculosis and non-Mycobacterium tuberculosis, It is possible to diagnose the type of non-mycobacteria simultaneously and to complete the present invention.

It is an object of the present invention to provide a primer set for multiplex PCR for simultaneous detection of Mycobacterium tuberculosis and non-mycobacteria.

Yet another object of the present invention is to provide a composition for simultaneous detection of Mycobacterium tuberculosis and non-Mycobacterium tuberculosis including a primer set for multiplex PCR for simultaneous detection of Mycobacterium tuberculosis and non-tuberculosis, and a kit comprising the composition.

Yet another object of the present invention is to provide a method for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis.

In order to accomplish the above object, the present invention provides a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: rv0577 A primer set for detection; A primer set for detection of RD9 (Region of Difference 9) comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; Primer sets for detection mtbk _20680 comprising the nucleotide sequence represented by nucleotide sequence SEQ ID NO: 8 and represented by SEQ ID NO: 7; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; ( Ocu-18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: A primer set for detection; And a primer set for detection of mab_3265c comprising the nucleotide sequence shown in SEQ ID NO: 13 and the nucleotide sequence shown in SEQ ID NO: 14, and a primer set for detecting mab_3265c for the simultaneous detection of mycobacteria and non-mycobacteria A primer set for multiplex PCR is provided.

The present invention also provides a primer set for detecting rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4; A primer set for RD9 detection comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; Primer sets for detection mtbk _20680 comprising the nucleotide sequence represented by nucleotide sequence SEQ ID NO: 8 and represented by SEQ ID NO: 7; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And a base sequence and a primer set for mab _3265c detection comprising the nucleotide sequence shown in SEQ ID NO: 14 shown in SEQ ID NO: 13; a, M. tuberculosis and non-tuberculosis co-detection comprising at least four kinds of primer sets selected from the group consisting of ≪ / RTI >

The present invention also provides a kit for detecting Mycobacterium tuberculosis and non-mycobacteria, comprising a composition for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis.

(A) a primer set for detection of rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4 in a sample ; A primer set for RD9 detection comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; Primer sets for detection mtbk _20680 comprising the nucleotide sequence represented by nucleotide sequence SEQ ID NO: 8 and represented by SEQ ID NO: 7; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And the primer set for detection mab _3265c comprising a base sequence shown in the nucleotide sequence and SEQ ID NO: 14 shown in SEQ ID NO: 13; the addition of at least four kinds primer set selected from the group consisting of performing a PCR; And (b) detecting mycobacteria and non-mycobacteria in the PCR product.

The use of the primer set for simultaneous detection of Mycobacterium tuberculosis and non-Mycobacterium according to the present invention can effectively discriminate the Beijing family, which is a worldwide prevalent Mycobacterium tuberculosis, and can distinguish clinically significant non-tuberculosis (NTM) and Mycobacterium tuberculosis (MTBC) And the type can be identified.

1 shows the results of MTB ( M. tuberculosis Beijing and M. tuberculosis non-Beijing) and NTM (MAH; M. avium subsp. Hominissius , MI; M. intracellulare , MAB; M. abscessus subsp. Abscessus and MAS; M. abscessus subsp . Massiliense and other mycobacteria using multiplex primer sets of the present invention.
FIG. 2 is a diagram showing the results of performing multiplex PCR on the MTB ( M. tuberculosis Beijing and M. tuberculosis non-Beijing) clinical strains.
FIG. 3 is a diagram showing the results of performing multiplex PCR on MTBC and NTM strains.
FIG. 4 shows the result of confirming the sensitivity of multiplex PCR according to the concentration of template DNA.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set for detecting rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4; A primer set for detection of RD9 (Region of Difference 9) comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; Primer sets for detection mtbk _20680 comprising the nucleotide sequence represented by nucleotide sequence SEQ ID NO: 8 and represented by SEQ ID NO: 7; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And SEQ ID NO: _3265 mab comprising a base sequence shown in the nucleotide sequence and SEQ ID NO: 14 represented by the 13 c A primer set for multiplex PCR for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis, which comprises at least four kinds of primer sets selected from the group consisting of a primer set for detection.

The primer set for multiplex PCR for simultaneous detection of Mycobacterium tuberculosis and non-mycobacteria of the present invention comprises a primer set for detecting 16S rRNA comprising the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2, And a primer set for multiplex PCR for simultaneous detection of non-Mycobacterium tuberculosis.

In the present invention, Region of Difference 9 (RD9) It means the part extending from rv2072c to rv2073c .

The term "detection" in the present invention means discriminating between Mycobacterium tuberculosis and non Mycobacterium tuberculosis by PCR using the primer set of the present invention, identifying the types of Mycobacterium tuberculosis and non Mycobacterium tuberculosis, and distinguishing between the Beijing family and the non-Beijing family .

The term "primer" in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a template of a complementary nucleic acid, Lt; RTI ID = 0.0 > nucleic acid < / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

In the present invention, the term " multiplex PCR "refers to a method of amplifying a plurality of target genes in the same reaction solution by simultaneously using a plurality of primer pairs.

Using the primer set of the present invention when using a single PCR reaction, unlike the traditional methods of PCR 16S rRNA, rv0577, RD9, mtbk _20680, IS 1311, DT1 (ocu_18960), can be amplified at the same time because the mab _3265 c It is possible to effectively distinguish the Beijing family, which is a worldwide prevalent type of tuberculosis, and at the same time to identify and differentiate between clinically significant non-tuberculosis (NTM) and tuberculosis (MTBC).

In the design of the primer, there are various restrictions such as a ratio of A, G, C and T content of the primer, prevention of formation of a primer complex (dimer) and repetition of the same base sequence three times or more. the conditions such as the amount of template DNA, the concentration of the primer, the concentration of dNTP, the concentration of Mg ^{2 +} , the reaction temperature, and the reaction time should be appropriate.

Further, in the multiplex PCR reaction in which two or more primer pairs are mixed and simultaneously subjected to PCR as in the present invention, at least two genes in the target sample are confirmed at one time, the same conditions as in the case of the single PCR in the primer design are more strict And the size of the gene product must be discriminated in order to distinguish the gene product amplified on the gel after completion of the reaction. In setting the reaction conditions, since a common reaction condition for all of the primers to be PCR should be set at once, a very difficult condition setting process is required as compared with a single PCR.

Such primers may incorporate additional features that do not alter the basic properties. I.e. the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more nucleotides into nucleotides, and uncharged linkages such as phosphonates, phosphotriesters, phosphoramidates or carbamates, phosphorothioates or phosphorodithioates Or the like can be transformed into a charged nucleus. The nucleic acid may be at least one of a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalating agent such as acridine or psoralen, a chelating agent such as a metal, a radioactive metal, And may have additional covalently bonded moieties.

The primer sequences of the present invention can also be modified using a label that can directly or indirectly provide a detectable signal. The primers can include labels that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful markers include ³² P, fluorescent dyes, electron dense reagents, enzymes (that is generally used in a ELISA), biotin, or haptens and proteins or antiserum possible the monoclonal antibodies used.

The primers of the present invention can be prepared by cloning and restriction cleavage of appropriate sequences and digestion with restriction enzymes such as the phosphotriester method of Narang et al. (1979, Meth, Enzymol. 68: 90-99), diethylphosphoramidate Including, for example, direct chemical synthesis methods, such as the solid support method of Tetrahedron Lett. 22: 1859-1862 (1981), and the solid support method of US 4458066.

In the present invention, " Mycobacterium tuberculosis (MTB)" means a pathogenic bacterium causing tuberculosis, "non-tuberculosis" means a tuberculosis non-tuberculous mycobacteria do.

In the present invention M. tuberculosis may be the M. tuberculosis Beijing family (MTB Beijing) or M. tuberculosis non-Beijing family (MTB non-Beijing), M is a non-TB in the present invention avium subsp. hominissuis (MAH), M. intracellulare (MI), M. abscessus subsp. abscessus (MAB), M. abcessus subsp. and massiliense (MAS).

The present invention also provides a primer set for detecting rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4; A primer set for RD9 detection comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; Primer sets for detection mtbk _20680 comprising the nucleotide sequence represented by nucleotide sequence SEQ ID NO: 8 and represented by SEQ ID NO: 7; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And the nucleotide sequence shown in SEQ ID NO: 13 and the nucleotide sequence shown in SEQ ID NO: mab _3265 c A primer set for detection, and a set of at least four primers selected from the group consisting of a primer set for detecting a mycobacteria and a non-mycobacteria.

The composition of the present invention may further comprise a primer set for detecting 16S rRNA comprising the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO:

The composition for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis of the present invention may further comprise a reaction amplification mixture, wherein the reaction amplification mixture is a reagent necessary for performing an amplification reaction, a thermostable DNA polymerase, a deoxynucleotide, Sterile water, and a solution containing a divalent metal cation, and the like, and may preferably include a reaction buffer, a deoxynucleotide, and a DNA polymerase.

In addition, the present invention provides a kit for simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis comprising the composition for simultaneous detection of Mycobacterium tuberculosis and non-tuberculosis.

In addition to the composition for simultaneous detection of nucleic acid and non-mycobacteria, the kit of the present invention may further comprise a reagent necessary for carrying out an amplification reaction, a thermostable DNA polymerase, a deoxynucleotide, a sterile water without nucleases and a solution containing a divalent metal cation , Preferably a reaction buffer, a deoxynucleotide, and a DNA polymerase.

(A) a primer set for detection of rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4 in a sample ; A primer set for RD9 detection comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; Primer sets for detection mtbk _20680 comprising the nucleotide sequence represented by nucleotide sequence SEQ ID NO: 8 and represented by SEQ ID NO: 7; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And SEQ ID NO: _3265 mab comprising a base sequence shown in the nucleotide sequence and SEQ ID NO: 14 represented by the 13 c A primer set for detection; and performing PCR by adding at least four sets of primers selected from the group consisting of primers set for detection; And (b) detecting mycobacteria and non-mycobacteria in the PCR product.

The method for detecting Mycobacterium tuberculosis and non-mycobacteria at the same time according to the present invention may further comprise the step of (c) identifying the type of Mycobacterium tuberculosis and mycobacteria in the step (b) in the PCR product.

In the present invention, the sample may be DNA isolated from Mycobacterium tuberculosis or non Mycobacterium tuberculosis, but the present invention is not limited thereto.

In carrying out the simultaneous detection method of Mycobacterium tuberculosis and non-mycobacteria according to the present invention, it is possible to carry out the reaction at a sample temperature, a dNTP concentration and a suitable temperature obvious to those skilled in the art. Although not limited thereto, it is preferable to use 1 μl of cDNA or PCR buffer solution in 25 μl of the PCR reaction solution.

In PCR, denaturation at 95 ° C for 10 minutes, denaturation at 96 ° C for 45 seconds, annealing at 60.5 ° C for 45 seconds, and extension of the sequence at 72 ° C for 40 seconds And the cycle is repeated 32 times and finally a final elongation is performed at 72 < 0 > C for 10 minutes. However, if the annealing temperature is maintained at 60.5 ° C, the remaining temperature can be adjusted to an appropriate level for a person skilled in the art as long as it is suitable for carrying out the PCR reaction.

The multiplex PCR is performed to amplify a gene present in the sample, and the amplified gene is electrophoresed on an acrylamide gel or an agarose gel to identify a band.

The method of simultaneous detection of Mycobacterium tuberculosis and Mycobacterium tuberculosis according to the present invention has an advantage of high sensitivity and specificity since the gene can be amplified from the sample at a level of 1,2 g / μl to 1 ng / μl.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1: Design of a primer

Based on the specific gene sequence of each species in six species of antibiotics causing major lung diseases in Korea, a primer set was designed to have at least 50 bp difference in amplified products. The six bacteria used in the preparation of the primer set are as follows.

1. M. tuberculosis Beijing family (MTB Beijing)

2. M. tuberculosis non-Beijing family (MTB non-Beijing)

3. M avium subsp. hominissuis (MAH)

4. M. intracellulare (MI)

5. M. abscessus subsp. abscessus (MAB)

6. M. abcessus subsp. massiliense (MAS)

The information of the primer set produced in the present invention is shown in Table 1.

The 16s rRNA gene is detectable in all mycobacteria, and the size of the amplified product is 506 bp.

rv0577 (NCBI Reference Sequence No. NP_215091) is detectable in all MTBC ( Mycobacterium tuberculosis complex) and the size of the amplified product is 705 bp.

RD9 (Genbank accession No. Y18604) can be detected in all MTB (Mycobacterium tuberculosis), designed to amplify a portion spanning from the rv2072 rv2073 c c and the size of the amplification product is 369 bp.

mtbk _20680 (Genbank accession No. AIB48613) can be detected specifically in MTB of M. tuberculosis Beijing family, the amount of amplification product is 231 bp.

IS 1311 (Genbank accession No. U16276) was obtained from MAC ( Mycobacterium avium (Insertion sequence, IS), which can be detected in all MAC except MI, and the size of the amplified product is 600 bp.

DT1 ( ocu_18960, Genbank accession No. L04543) can be detected in M. intracellulare , and the size of the amplified product is 106 bp.

mab _ 3265c (Genbank accession No. CAM63341.1 ) has got to the gene present in the MAS, the MAB mass_3210 (Genbank accession No. AGM29812.1) gene is inserted between mab _3265c with mab _ 3266c gene amplification with different size Specific amplification, and the size of each amplification product is 310 bp and 1145 bp.

Example 2: Preparation of DNA samples for PCR

Genomic DNA was extracted from the strain of Example 1 by modifying the method of Murray and Thomson (1980) in order to obtain a DNA sample to be used in the PCR of the present invention. Specifically, the strain was inoculated into 10 ml of a medium broth containing 10% (v / v) OADC in Middlebrook 7H9, and cultured at 37 ° C and 200 rpm for 2-4 weeks to sufficiently extract the DNA. The cultured bacteria were boiled at 80 ° C for 30 minutes and inactivated, and centrifuged at 4000 rpm for 10 minutes. The collected precipitates were suspended in 300 μl of 1 × TE buffer in a microcentrifuge tube. 100 [mu] l of lysozyme was added thereto at a concentration of 100 [mu] g / ml, and the mixture was allowed to stand at 37 [deg.] C for 2 days. 30 μl of proteinase K and 100 μl of 10% (w / v) SDS at a concentration of 20 mg / ml were allowed to stand at 65 ° C for 3 hours. 100 ㎕ of 5 M NaCl was added to the mixed solution, and 80 의 of hexadecyltrimethyl ammonium bromide (CTAB) / NaCl was left at 65 캜 for 10 minutes and 20 minutes, respectively. The supernatant obtained by adding 700 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v) solution and centrifuging at 13000 rpm for 20 minutes at 4 ° C was taken and the same procedure was repeated once And repeated. 0.6 parts of isopropyl acetate was added to the solution thus obtained and mixed, followed by stabilization at -20 占 폚 for 1 hour. After stabilization, the mixture was centrifuged at 13,000 rpm for 1 minute at 13000 rpm, and then 200 μl of 1 × TE buffer containing RNase was added to the precipitate. The mixture was allowed to stand at 65 ° C for 1 hour and then stored at 4 ° C. This was used as template DNA for PCR.

Example 3: Establishment of PCR reaction conditions

Mycobacterium tuberculosis HN878 (Beijing family), Mycobacterium tuberculosis H37Rv (Non-Beijing family), Mycobacterium bovis BCG, Mycobacterium avium subsp. hominissuis , Mycobacterium intracellulare , Mycobacterium abscessus subsp. abscessus , Mycobacterium abscessus subsp. massiliense , Mycobacterium kansasii , Escherichia The DNA was extracted from E. coli according to the same method as in Example 2. Template DNA 1 ㎕, 2X EF-Taq PCR Smart mix2 (. SolGent co, Ltd., Daejeon, South Korea) 25 ㎕, each primer set of 10 pmole [mtbk _20680, DT1 ( ocu_18960): 16s rRNA gene, RD9 (rv2072 c ~ rv2073 c), iS 1311 , mab _3265 c: rv0577 0.6 ratio of the primer sets: 1 such that the 4 (㎕)] were mixed and then the PCR reaction mixture was added to sterile distilled water to a final volume to 50 ㎕ the .

The reaction mixture was amplified with a S1000 Thermal Cycler (BIORAD) machine at 95 ° C. for 10 minutes, at 96 ° C. for 45 seconds, at 60.5 ° C. for 45 seconds, at 72 ° C. for 40 seconds at 32 ° C. and at 72 ° C. for 10 minutes. The PCR amplification products were electrophoresed on a 6% (v / v) acrylamide DNA gel for 1 hour and the results are shown in FIG.

As shown in Figure 1, MTB Beijing family sample the 16s rRNA (506 bp), rv0577 (705 bp), RD9 (rv2072 c ~ rv2073 c, 369 bp), mtbk _20680 (231 bp) band appeared, MTB ratio - In the Beijing family sample, 16s rRNA (506 bp), rv0577 , RD9 ( rv2072 c to rv2073 c) 16s rRNA (506 bp) and IS 1311 (600 bp) bands were observed in the MAC samples and 16s rRNA (506 bp) and DT1 ( ocu_18960, 106 bp) band appeared, MAB is a 16s rRNA (506 bp), mab _3265 c (310 bp) was a band that appeared, MAS is a 16s rRNA (506 bp), mab _3265 c (1145 bp) band appears Respectively. In the negative control E. coli , no band was observed.

therefore, The composition containing the primer set of the present invention showed different band patterns depending on the type of strain, and thus it was confirmed that the composition can be analyzed for major lung diseases including MTB Beijing family.

Example 4: Identification of specificity and sensitivity of primer

4.1 Primer  Identification of specificity

 In order to confirm the specificity of the primer set of the present invention, PCR was performed on 69 kinds of mycobacterial DNAs listed in Table 2 below.

Template DNA was extracted from mycobacteria in Table 2 by the method of Murray and Thomson (1980), and PCR was performed in the same manner as in Example 3 above. FIG. 2 shows the results of multiplex PCR for MTB (clinical isolates of M. tuberculosis and non-Beijing family), and FIG. 3 shows results of multiplex PCR of MTBC and NTM strains.

As shown in FIG. 2 and FIG. 3, a gene amplification product specific to the major lung disease antioxidant as the target was confirmed, and only the 16s rRNA (506 bp) amplified product was confirmed for the strains other than the lung disease antioxidant. In addition, since no amplification products are formed in microorganisms other than antioxidants, it has been confirmed that when multiplex PCR is performed using the primer set of the present invention, major lung disease antioxidants can be specifically and rapidly detected.

4.2 Primer  Sensitivity check

In order to confirm the sensitivity of the primer set of the present invention, the DNA extracted by the CTAB extraction method from Mycobacterium tuberculosis HN878 strain was used. The DNA concentration was measured by the absorbance measurement method (260 nm), and the DNA was determined to be 1200 ng / The concentration was adjusted. The adjusted DNA was diluted in a total of 9 steps from 1200 g / 부터 to 0.1 ng / 희 by dilution and PCR was performed. PCR was carried out by 1 [mu] l of each of diluted DNA, and the result is shown in Fig.

As shown in FIG. 4, PCR was performed on the diluted DNA, and it was confirmed that gene amplification products were generated up to 1 ng / μl level.

Therefore, when the primer set of the present invention is used, it is confirmed that it is possible to detect the major lung disease antioxidant bacteria because the sensitivity is excellent.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A multiplex PCR primer set, a composition and a kit          the same for diagnosing Mycobacterium tuberculosis and          비 tuberculous mycobacteria <130> 1_20P <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for 16S rRNA <400> 1 gagatactcg agtggcgaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for 16S rRNA <400> 2 caacgcgaca aaccacctac 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rv0577 <400> 3 atgcccaaga gaagcgaata ca 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rv0577 <400> 4 aatgtcagcc ggttccgcaa 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for RD9 <400> 5 gtgtaggtca gccccatcc 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for RD9 <400> 6 gtaagcgcgt ggtgtgga 18 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mtbk_20680 <400> 7 ttatgccaga aatacacccg cg 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mtbk_20680 <400> 8 aatcgcgggc ttgtggctac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IS1311 <400> 9 tcgatcagtg cttgttcgcg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IS1311 <400> 10 cgatggtgtc gagttgctct 20 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DT1 <400> 11 aaggtgagcc cagctttgaa ctcca 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DT1 <400> 12 gcgcttcatt cgcgatcatc aggtg 25 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mab_3265c <400> 13 gcttgttccc ggtgccacac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mab_3265c <400> 14 ggagcgcgat gcgtcaggac 20

Claims (8)

(a) a primer set for detection of rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4;
(b) a primer set for detection of mtbk_20680 comprising the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 8;
(c) a primer set for detecting 16S rRNA comprising the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2; And
(d) a primer set for detection of RD9 (Region of Difference 9) comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And a primer set for detection of mab - 3265c comprising the nucleotide sequence shown in SEQ ID NO: 13 and the nucleotide sequence shown in SEQ ID NO: 14; and a set of two or more primers selected from the group consisting of:
A primer set for multiplex PCR for simultaneous detection and discrimination of Mycobacterium tuberculosis and non Mycobacterium tuberculosis.
delete The method according to claim 1, wherein the Mycobacterium tuberculosis is selected from the group consisting of M. tuberculosis MTB Beijing family or M. tuberculosis MTB non-Beijing family for multiplex PCR for simultaneous detection and discrimination of Mycobacterium tuberculosis and non- Primer set.
The method of claim 1, wherein the non-Mycobacterium tuberculosis is M. avium subsp. hominissuis (MAH), M. intracellulare (MI), M. abscessus subsp. abscessus (MAB), M. abcessus subsp. and massiliense (MAS). A primer set for multiplex PCR for simultaneous detection and discrimination of Mycobacterium tuberculosis and non-mycobacteria.
(a) a primer set for detection of rv0577 comprising the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4;
(b) a primer set for detection of mtbk_20680 comprising the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 8;
(c) a primer set for detecting 16S rRNA comprising the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2; And
(d) a primer set for detection of RD9 (Region of Difference 9) comprising the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 6; A primer set for detecting IS 1311 comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10; A primer set for detecting DT1 ( ocu_18960 ) comprising the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12; And a primer set for detection of mab - 3265c comprising the nucleotide sequence shown in SEQ ID NO: 13 and the nucleotide sequence shown in SEQ ID NO: 14; and a set of two or more primers selected from the group consisting of:
Composition for simultaneous detection and discrimination of Mycobacterium tuberculosis and non Mycobacteria.
A kit for simultaneous detection and discrimination of mycobacteria and non-mycobacteria comprising the composition of claim 5.
(a) performing PCR by adding the primer set of claim 1; And
(b) detecting mycobacteria and non-mycobacteria in the PCR product.
The method according to claim 7, further comprising (c) identifying the type of Mycobacterium tuberculosis and mycobacteria in the step (b) in the PCR product.

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