JPH09103300A - Oligonucleotide for amplifying beta-subunit gene in polymerase for bacillus tuberculosis rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel nucleic acid which includes a specific DNA sequence or a part thereof, is used in the amplification of the β-subunit gene of Bacillus tuberculosis RNA polymerase and is useful for detection of chemical tolerance, an important index for selecting the therapeutic agent for tuberculosis. SOLUTION: This novel oligonucleotide includes nucleic acid sequence of at least 15 sequential residues selected from the nucleic acid sequences given in formula I or formula II or their complementary DNA sequences and is used in amplification of the β-subunit gene of Mycobacterium tuberculosis. The gene fragment of RNA polymerase β-subunit containing the area causing the mutation obtaining resistance to rifampicin in high frequency is specifically, simply, rapidly and steadily amplified and is useful in the examination on the drug resistance. This oligonucleotide may be prepared on the basis of gene information on Escherichia coli or the like relating to chemical resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to clinical diagnosis,
Oligonucleotide capable of specifically amplifying an RNA polymerase β subunit gene fragment containing a region in which a mutation involved in acquisition of resistance to rifampicin of Mycobacterium tuberculosis occurs frequently, its use, and the above oligonucleotide The present invention relates to a method of using a DNA fragment amplified by using.

[0002]

2. Description of the Related Art Mycobacterium tuberculosis
Is a Gram-positive bacillus that has anti-acidic properties and is a pathogen that causes serious diseases such as tuberculosis. Pulmonary tuberculosis is by far the most common tuberculosis disease. Mycobacterium tuberculosis causes tuberculous lesions in almost all organs, including tuberculous pleurisy, tuberculous meningitis, caries, intestinal tuberculosis, renal tuberculosis, and joint tuberculosis. The source of infection is the patient, which is transmitted through the respiratory tract by droplet infection or the like.

In recent years, the anti-tuberculosis drug rifampicin has been used for the treatment of tuberculosis, and the cure rate for tuberculosis disease tends to increase. Rifampicin acts on the RNA polymerase β subunit of Mycobacterium tuberculosis, suppresses its action, and inhibits the growth of Mycobacterium tuberculosis, thereby exhibiting antibacterial properties. However, when a part of the RNA polymerase β subunit is mutated and rifampicin cannot act, the tubercle bacillus becomes resistant to rifampicin. Such rifampicin-resistant bacteria make treatment of tuberculosis disease difficult. Prompt detection of the resistance of infected M. tuberculosis to rifampicin can be an important indicator in selecting therapeutic agents. Currently, drug resistance tests of tubercle bacilli such as rifampicin are conducted by a culture method. However, Mycobacterium tuberculosis generally grows slowly and requires a considerable amount of time for cultivation. Usually, the direct method requires 4 to 8 weeks, and the indirect method requires 8 to 16 weeks. Therefore, a rapid method for detecting resistance to rifampicin is desired.

Rifampicin-resistant Mycobacterium tuberculosis has about 69 genes in the gene encoding the RNA polymerase β subunit.
It has been reported to have mutations accompanied by amino acid mutations in base pairs (Lancet; 341: 647, 1993).
Year, Antimicrobial Agents and Chemotherapy; Vol. 37, 1
0, page 2054, 1993, J. Cliical Microbiolog
y; 32, p. 1095, 1994). It is known that the mutation that confers resistance to rifampicin to Mycobacterium tuberculosis is not limited to the mutation of a specific amino acid, but is widely distributed in this 69 base pair region.

Based on these research results, a small amount of RNA polymerase β subunit gene fragment related to resistance to rifampicin of Mycobacterium tuberculosis present in clinical specimens is
The development of a method for estimating the presence or absence of rifampicin resistance based on the gene mutation in the fragment amplified by PCR (polymerase chain reaction) has attracted attention. Mutations can be detected by direct sequence method, SSCP
(Single-strand conformation polymorphism) method, hybridization method using an oligonucleotide probe, RFLP method and the like are considered. By using these methods, it becomes possible to estimate the presence or absence of resistance of M. tuberculosis to rifampicin within about 2 days.

However, DNA extracted from clinical specimens
Often contains DNAs derived from various microorganisms other than Mycobacterium tuberculosis, so it is necessary to specifically amplify and analyze only the gene of Mycobacterium tuberculosis. In particular, bacteria belonging to the genus Mycobacterium other than Mycobacterium tuberculosis have high homology in gene sequences, and it is necessary to fully consider the possibility of simultaneous amplification.

Diseases caused by Mycobacterium other than Mycobacterium tuberculosis are called atypical mycobacteriosis,
It is known that it may show symptoms similar to tuberculosis. Among them, the atypical mycobacteriosis caused by Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellulare, etc. has a high frequency. Moreover, many non-pathogenic bacteria of the genus Mycobacterium have been isolated from various clinical materials. This suggests that various Mycobacterium may be contained in the clinical specimen. In addition, there is a report that some acid-fast bacterium DNA was detected in distilled water for analysis, etc., and attention must be paid to contamination from the environment.

[0008] Oligonucleotides for amplifying the RNA polymerase β subunit gene of Mycobacterium tuberculosis reported so far (Lancet; 341: 647, 1993, Antimicr
obial Agents and Chemotherapy; Vol. 37, No. 10, 205
P. 4, 1993, J. Cliical Microbiology; 32 Volume 10
(P. 95, 1994) uses sequences that are relatively conserved among Mycobacterium species, so it is clear that at least some Mycobacterium bacterium genes are amplified as well. In fact, when amplification is performed using the reported sequence, amplification of the target DNA can be confirmed even in some major atypical mycobacteria such as Mycobacterium kansasii. It has been confirmed that the RNA polymerase β subunit genes other than Mycobacterium tuberculosis thus amplified contain at least some gene sequences different from those of Mycobacterium tuberculosis, which makes determination difficult when analyzing clinical samples. Cases are expected to occur. With these things in mind,
When a clinical sample is used as a material, it is considered very important to perform analysis using a DNA fragment obtained by amplification using an oligonucleotide highly specific to Mycobacterium tuberculosis, particularly in high-sensitivity analysis.

[0009]

The object of the present invention is to specifically target an RNA polymerase β subunit gene fragment containing a region in which a mutation involved in acquisition of resistance to rifampicin of Mycobacterium tuberculosis occurs frequently. It is intended to provide a novel oligonucleotide for amplification, its use, and a method of using a DNA fragment amplified using the above-mentioned oligonucleotide.

[0010]

[Means for Solving the Problems] As a result of repeated studies on the RNA polymerase β subunit genes of Mycobacterium tuberculosis and other Mycobacterium, the present inventors have found that they are almost specific to Mycobacterium tuberculosis. The present invention has been completed by obtaining a nucleic acid sequence, establishing a gene amplification method using the nucleic acid sequence.

The present invention comprises Mycobacterium tuberculosis RNA containing a nucleic acid sequence consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 or 2 or the nucleic acid sequences complementary to the sequences. This is an oligonucleotide for amplifying a polymerase β subunit gene. The above oligonucleotide may be modified with a modified product. This modified product is preferably a radioisotope, a fluorescent substance, a luminescent substance, biotin, or digoxigenin.

The present invention is also a method for amplifying the Mycobacterium tuberculosis RNA polymerase β subunit gene using the above-mentioned oligonucleotide.

The present invention is also a method for amplifying nucleic acid in a sample using the above-mentioned oligonucleotide, and analyzing the presence or absence of base substitution in the DNA fragment using the obtained DNA fragment as a sample.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have already determined the sequences of Escherichia coli, Pseudomonas bacteria, Mycobacterium bacteria, etc., and some mycobacteria revealed by the present inventors. R of Umbrella
As a result of repeated theoretical and experimental verification of homology of gene sequences among various species with reference to the sequence of the NA polymerase β subunit gene, Mycobacterium tuberculosis (Mycobacterium tuberculosis)
We have found specific oligonucleotide sequences (SEQ ID NOS: 1 and 2) for amplifying an RNA polymerase β subunit gene fragment containing a region in which a mutation involved in acquisition of rifampicin resistance of rculosis) occurs frequently.

That is, the present invention relates to Mycobacteria
It is an oligonucleotide that specifically amplifies an RNA polymerase β subunit gene fragment containing a region in which mutations involved in acquisition of resistance to rifampicin of (ium tuberculosis) occur frequently.

The above-mentioned oligonucleotide is a nucleic acid sequence set forth in SEQ ID NO: 1 or 2 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is uracil (U). ) May be substituted)
Or a part or all of their complementary sequences.

The oligonucleotides of the present invention can be used as primers for DNA amplification. The oligonucleotides of the present invention can also be used as primers when performing sequence analysis. The oligonucleotide need not use the entire sequence of SEQ ID NO: 1 or 2 or their complementary sequences. Sequences of an appropriate length derived from the sequences in the sequence listing and their complementary sequences are
It can be determined by calculating the dissociation temperature (Tm value) according to the R reaction conditions and the like. However, in order for the primer to have sufficient specificity, a length of at least 15 contiguous bases, more preferably at least 20 contiguous bases, is required. In order for the primer to have sufficient specificity, it is desirable to use the 3'-terminal base described in the sequence in the sequence listing as the 3'-terminal base. It can be easily predicted by those skilled in the art that some substitutions may be made in the oligonucleotide depending on the conditions to be used, the purpose and the like. Further, it can be easily expected to further extend the 5 ′ side of the primer according to the sequence of Mycobacterium tuberculosis so as not to affect the specificity of the primer.

In designing the oligonucleotide, the present inventors consider that there is also a mutant in which the base in the target gene complementary to the 3'end of the oligonucleotide is mutated, and the probability of mutation is low. The base was set as the base at the 3'end of the oligonucleotide. The base at the 3'end of the oligonucleotide that serves as a polymerase primer serves as the starting point of the polymerase reaction. Therefore, if the sequence of the target gene is mutated,
The efficiency of the polymerase reaction will be extremely reduced.
In fact, 3'of the oligonucleotide shown in SEQ ID NO: 1
When the T (plus chain) of the Mycobacterium tuberculosis gene corresponding to the terminal base (T) is mutated to A or G, leucine encoded by TTG is translated as methionine (ATG) or valine (GTG). Moreover, when A (plus chain) of the Mycobacterium tuberculosis gene complementary to the 3'end (T) of the oligonucleotide shown in SEQ ID NO: 2 is mutated to C or T, the glutamic acid encoded by GAA is aspartic acid (GAC). Or GAT). Leucine and GA encoded by this TTG
The glutamic acid encoded by A is almost completely conserved across species at the amino acid level. This means that these amino acids play an important role in maintaining the function of the protein, and when these amino acids are mutated to other amino acids by mutation, the clone is likely to disappear by natural selection. It means that. Therefore, it can be considered that almost all the RNA polymerase β subunit genes derived from M. tuberculosis that can exist can be universally amplified by the amplification using the above-mentioned oligonucleotide.

The above-mentioned oligonucleotide can be modified with a modified product. This modified product is preferably a known label such as a radioactive substance, a fluorescent substance, a luminescent substance, biotin and digoxigenin. The form of labeling may be either end labeling or labeling in the middle of the sequence.

The present invention includes a method for amplifying Mycobacterium tuberculosis RNA polymerase β subunit gene using the above-mentioned oligonucleotide.

The present invention also includes a method of amplifying nucleic acid in a sample using the above-mentioned oligonucleotide, and using the obtained DNA fragment as a sample to analyze the presence or absence of base substitution in this DNA fragment.

In the present invention, the target of DNA amplification is DNA derived from Mycobacterium tuberculosis in various clinical materials such as sputum, lung lavage fluid, pleural effusion, gastric juice and blood. It goes without saying that amplification is also included. Further, the sample in the present invention includes the above-mentioned various clinical materials and cultured bacterial cells, as well as nucleic acids isolated and purified from these.

Primers that can be used for DNA amplification include all the oligonucleotides described above.
A PCR (polymerase chain reaction) method can be generally used for this DNA amplification. As described above, the combination of the oligonucleotides shown in SEQ ID NOS: 1 and 2 can be preferably used for the amplification of Mycobacterium tuberculosis RNA polymerase β subunit from a clinical sample. However, by further reamplifying the DNA amplified by the primers outside the oligonucleotides shown in SEQ ID NOS: 1 and 2 with the oligonucleotides shown in SEQ ID NOS: 1 and 2, respectively, a higher sensitivity can be obtained. It is expected that amplification will be possible. The outer primer used at this time is preferably specific to Mycobacterium tuberculosis, but a primer having low specificity can also be used.

In amplification, SEQ ID NOS: 1 and 2
It is important to use the combination of oligonucleotides shown in each. This is because the specificity of the amplification reaction depends on the synergistic effect of the two oligonucleotides used. Further, in order to further increase the specificity, it is important to keep the concentration of the primer used for amplification as low as possible.

A method for amplifying a region of the RNA polymerase β subunit involved in resistance to rifampicin, which has been reported so far (Lancet; 341: 647, 199).
3rd year, Antimicrobial Agents and Chemotherapy; 37
Volume 10, page 2054, 1993, J. Cliical Microbiol
ogy; 32: 1095, 1994) has low specificity for M. tuberculosis because it performs PCR amplification using an oligonucleotide having a sequence quite common to the genus Mycobacterium as a primer. In particular, when the RNA polymerase gene of Mycobacterium tuberculosis is directly amplified from a clinical sample (sputum, etc.), genes of other Mycobacterium bacteria may also be amplified. This must be taken into consideration when applying it in the clinical setting. INDUSTRIAL APPLICABILITY The present invention makes it possible to specifically amplify a region related to rifampicin resistance of the RNA polymerase β subunit gene of Mycobacterium tuberculosis, and has solved the problem when using a clinical sample or the like.

The present inventors have used the oligonucleotides having the sequences shown in SEQ ID NOS: 1 and 2 as primers to prepare Mycobacterium tuberculos.
It was experimentally confirmed that an RNA polymerase β subunit gene fragment containing a region in which a mutation involved in the acquisition of rifampicin resistance of (is) frequently occurs can be specifically amplified by PCR. In particular,
Using the combinations of oligonucleotides shown in SEQ ID NOS: 1 and 2 as primers, DNAs of 14 species of Mycobacterium were used as samples for PC.
An R amplification experiment was performed. As a result, Mycobacterium tuberculosis (Mycoba
cterium tuberculosis) and Mycobacterium vobis that are classified into the same group of Mycobacterium tuberculosis
Amplification of the A polymerase β subunit gene was confirmed. In addition, amplification of the target gene was not found in DNA of bacteria other than Mycobacterium, such as Escherichia coli and Staphylococcus aureus, which can be frequently isolated from clinical materials. Mycobacterium tuberculosis (Mycobacterium vobis) is a bacterium that is classified into the Mycobacterium tuberculosis group together with human tuberculosis (Mycobacterium tuberculosis), and has the same pathogenicity. It is known that M. bovis is very difficult to distinguish from human even in genotyping using a gene encoding rRNA, and the structure of the RNA polymerase β subunit gene is also M. tuberculosis. It is speculated that it is very similar to. However, the disease caused by Mycobacterium bovis is extremely rare, and it can be said that the possibility of contamination with clinical materials is extremely low.

The amplified DNA analysis method used in the present invention includes various analysis methods. Various analysis methods include direct sequence method, SSCP (Single-strand
conformation polymorphism) method, hybridization analysis using an oligonucleotide probe, and the like. Among them, the direct sequencing method is the most desirable method because it can detect all possible mutations. However, the direct sequence method is complicated in operation. When analyzing a large amount of samples at one time, it may be better to use a simple method such as SSCP method or hybridization analysis using an oligonucleotide.

The direct sequence method in the present invention is
In this method, a DNA fragment obtained by amplification is used as a template for sequence analysis by a nucleotide sequence determination method such as the dideoxy method. Since this method is simpler than the conventional method of cloning amplified DNA in a vector and analyzing it, it is more suitable for clinical examination. The direct sequencing method of the present invention is specifically a method of performing a sequence analysis using a DNA fragment obtained by amplification using the oligonucleotides shown in SEQ ID NOS: 1 and 2 of the present invention as a primer, as a template. Is. As a sequence primer, the oligonucleotides shown in SEQ ID NOS: 1 and 2 can be used depending on the application. Oligonucleotides with any of the sequences contained in the amplified sequence can also be used as primers. Furthermore, a primer modified with a modification such as a label can also be used. As a sign,
Radioisotope, fluorescent substance, luminescent substance, biotin,
Examples include digoxigenin. It can be seen that the sequencing primer is well used for sequence analysis with or without labeling. By using this method, all possible mutations can be analyzed. It is expected that this analysis may serve as a clear diagnostic criterion by clarifying the detailed relationship between the types of mutations of the above genes and the degree of resistance to rifampicin, that is, the minimum inhibitory concentration.

The SSCP method in the present invention is the DNA fragment obtained by amplification is heat-denatured into a single-stranded state,
This is a method of performing electrophoresis on a neutral acrylamide gel and determining the presence or absence of mutation from the difference in mobility (Genomics; Vol. 5, pp. 874, 1989). In particular,
The DNA fragments obtained by amplification using the oligonucleotides shown in SEQ ID NOS: 1 and 2 of the present invention as primers are heat-denatured into a single-stranded state, and subjected to strict temperature control under neutral acrylamide gel. In this method, electrophoresis is performed and the presence or absence of mutation in the RNA polymerase β subunit is determined from the difference in mobility. In the above SSCP method, by using an oligonucleotide modified with a modified product such as a label as a primer, more sensitive analysis can be performed. Examples of the label include radioisotopes, fluorescent substances, luminescent substances, biotin, digoxigenin and the like. By this method, the presence or absence of mutation can be detected more easily. However, this SSCP method cannot identify the position of the mutation. For this reason,
When a mutation is detected, it is necessary to analyze it by direct sequencing to identify the position of the mutation.

Hybridization analysis in the present invention means to hybridize a DNA fragment obtained by amplification with an oligonucleotide having various mutations,
This is a method of identifying a mutation site based on the presence or absence of hybridization. Specifically, the DNA fragments obtained by amplification using the oligonucleotides shown in SEQ ID NOS: 1 and 2 of the present invention as primers are hybridized with oligonucleotide probes containing various mutations, and the presence or absence of hybridization thereof is detected. This is a method for identifying mutations more. By this method, the mutation position can be rapidly identified. However, it is expected that it will be difficult to analyze all mutations. Therefore, hybridization analysis should be used, such as for some major types of selection.

By using the above methods alone or in combination, the conventional direct method takes 4 to 8 weeks and the indirect method gives 8 weeks.
The rifampicin resistance test, which took ~ 16 weeks, can be performed within about 2 days.

[0032]

The effects of the present invention will be further clarified by exemplifying embodiments of the present invention.

Example 1 (1) Synthesis of Oligonucleotides The oligonucleotides for amplifying Mycobacterium tuberculosis RNA polymerase β subunit were synthesized by a phosphoamidite method using a DNA synthesizer type 392 manufactured by ABI, and SEQ ID NO: 1 and It was carried out by synthesizing oligonucleotides having the sequences shown in 2 respectively. The procedure was according to the manual of ABI, and various oligonucleotides were deprotected with ammonia water at 55 ° C. overnight. HPL for purification
Using C (manufactured by Beckman), a reversed phase chromatography column (manufactured by Nakarai Tesque) was used.

(2) Preparation of sample DNA isolated from the bacteria shown below was used as a sample for PCR. All of the bacteria are clinical isolates, and after cultivation, the bacterial species have already been identified by the shape of colonies and various conventional biochemical tests. For the bacterium of the genus Mycobacterium, a colony (one platinum loop) on an Ogawa medium is prepared according to the method described in Kekkaku; Vol. 67, p. 795, 1992, and the final concentration is about 1 ng / μl in the PCR reaction solution. Used as. The quality of the extracted DNA was checked by confirming its amplification by a qualitative PCR test using a gene encoding rRNA as an amplification target. For other bacteria, extract each DNA
What was prepared according to a conventional purification method was used in a PCR reaction solution,
The final volume was about 1 ng / μl. These also confirmed the quality of DNA by PCR targeting other regions.

1. Mycobacterium tuberculosis 1. Mycobacterium tuberculosis 3. 3. Mycobacterium tuberculosis 4. 4. Mycobacterium tubercu1osis 5. Mycobacterium tuberculosis 6. 7. Mycobacterium tuberculosis 7. 7. Mycobacterium tuberculosis 8. Mycobacterium tuberculosis 9. Mycobacterium tuberculosis 10. Mycobacterium bovis 11. Mycobacterium kansashii
kansasii) 12. Mycobacterium malynum
rinum) 13. Mycobacterium siemia
miae) 14. Mycobacterium godonae
gordonae) 15. Mycobacterium sz
ulgai) 16. Mycobacterium avium
ium) 17. Mycobacterium intracellulare
terium intracellulare) 18. Mycobacterium Gustry
gastri) 19. Mycobacterium non-chromogenic
rium nonchromogenicu) 20. Mycobactidium fortuitum
erium fortuitum) 21. Mycobacteriu
m smegmatis) 22. Mycobacterium cheronei
elonei) 23. Staphylococcus aureus 24. Escherichia coli.

(3) PCR The oligonucleotides shown in SEQ ID NOS: 1 and 2 were
They were used as upstream and downstream primers for PCR, respectively. The composition of the reaction solution is 0.1 μM for each primer and 0.
15 mM of dATP, dCTP, dGTP, dTTP and Taq DNA polymerase (AmpliTaq: manufactured by Roche) 40 units / ml, and a 1-fold concentration attached buffer (manufactured by Roche). In actual use, 25 μl of the reaction composition solution having a double concentration was prepared, and an equal amount of the sample DNA solution was added to make 50 μl, which was then used in the following reaction.

The reaction conditions are as follows: Heat denaturation: 94 ° C., 20 seconds Annealing: 65 ° C., 20 seconds Extension reaction: 72 ° C., 45 seconds The above cycle was repeated 40 times. These operations are Perkin-Elmer / Cetu
s) DNA thermal cycler (GeneAmp9600R).

(4) Detection 10 μl of the reaction solution was electrophoresed on a 2% agarose gel and stained with ethidium bromide, and then fluorescence under UV irradiation was detected with a polaroid film. The electrophoresis conditions were a constant voltage of 100 V and 30 minutes. The procedure and other conditions are described in Maniatis et al. Molecular Cloning (1982
Year). In addition to the reaction solution, a molecular weight marker was also electrophoresed at the same time, and the length of the detected DNA fragment was calculated by comparing the relative mobilities.

(5) Results After agarose gel electrophoresis and ethidium bromide staining, fluorescence under UV irradiation was detected with a poradoid film, and a band of about 258 base pairs was confirmed. As a result, as shown in Table 1 below, target bands were confirmed in all 9 strains of Mycobacterium tuberculosis and Mycobacterium bovis. But,
No target band was confirmed in other Mycobacterium, Staphylococcus aureus, and Escherichia coli.

[0040]

[Table 1]

From the above, it was found that by using the oligonucleotide according to the present invention for PCR, the RNA polymerase β subunit gene fragment of human type and Mycobacterium bovis can be specifically amplified. From this, clinical specimens (sputum, sputum, etc.) containing Mycobacterium bacteria other than the Mycobacterium tuberculosis group and Staphylococcus aureus, Escherichia coli, etc.
Pleural effusion, lung lavage fluid, etc.) was found to be capable of specifically amplifying only the RNA polymerase β subunit gene fragment of Mycobacterium tuberculosis.

Example 2 (1) Extraction of DNA from clinical sputum 1 ml of the clinical sputum shown below was placed in a centrifuge tube, and an equal amount of NALC-NaOH (0.05 M trisodium citrate, 2
% Sodium hydroxide, 0.5% N-acetyl-L-cysteine)
Was added, and the mixture was stirred and then left at room temperature for 15 minutes. To the treated sample solution, 48 ml of 0.067 M phosphate buffer was added and mixed, and the mixture was centrifuged at 3,500 g for 15 minutes at room temperature. After centrifugation, the supernatant was removed by decantation, and the obtained precipitate was suspended in 1 ml of the above phosphate buffer solution to give a pretreatment solution.
For DNA extraction, use the Sputum specimen preparation kit (Roche) and follow the attached instructions to prepare 100 μl of pretreatment solution.
More extraction was performed. It was expected that the extract using this kit would still contain significant impurities, so phenol-chloroform treatment and ethanol precipitation were performed and the further purified DNA was used for the following amplification: Sputum 1 Low-concentration cases 2. Sputum 2 Low-concentration cases 3. Sputum 3 high-concentration cases.

(2) PCR The PCR was carried out under the same conditions as (3) of Example 1. At the same time, DNA and distilled water extracted from Mycobacterium tuberculosis (wild strain) were similarly amplified and used as a control.

(3) Detection It was carried out under the same conditions as (4) of Example 1.

(4) Results After completion of PCR, agarose gel electrophoresis was performed, and after staining with ethidium bromide, fluorescence under UV irradiation was detected on a polaroid film. As a result, a single band of about 258 base pairs was detected in all the samples examined this time (Fig. 1). From this, it was found that the RNA polymerase β subunit gene can be amplified from a clinical sample using the primer of the present invention. Sputum 1 and 2 are rRNA
It is a sample that has been proved to be a sample close to the detection limit by a PCR qualitative test targeting at. Therefore, it was proved that the amplification using the primer of the present invention can amplify the RNA polymerase β subunit gene of Mycobacterium tuberculosis from a clinical sample even when it is present at a low concentration in the clinical sample.

Example 3 (1) Purification of Amplified DNA To 40 μl of the PCR amplification solution obtained from the sample shown below in Example 2, an equal amount of 7.5M ammonium acetate and an equal amount of isopropanol were added, and the mixture was cooled to room temperature. After standing for 10 minutes at 12,000 rpm, it was centrifuged at room temperature for 15 minutes at room temperature. After removing the supernatant by decantation, 1 ml of 70% cold ethanol solution was added and mixed, and then 12,000 rp was added again.
Centrifuge at m for 5 minutes, remove the supernatant and dissolve the precipitate in 20 μl of distilled water: Mycobacterium tuberculosis (wild strain) 2. Sputum 1 3. Sputum 2 4. Sputum 3.

(2) Sequencing reaction / electrophoresis Sequence analysis of the DNA fragment purified in (1) was carried out by non-RI cycle sequencing kit Sequencing high-Plus- (manufactured by Toyobo) using biotinylated dideoxynucleotide and thermostable polymerase. ) Was used. As the sequence primer, the oligonucleotide of SEQ ID NO: 1 was used, and it was prepared to be 0.2 pmol / μl in the reaction solution. In addition, the concentrations of other reaction compositions (deoxynucleotide, ΔTth DNA polymerase, reaction buffer) were in accordance with the kit attachment. 4 μl of the DNA solution prepared in (1) was added to 13 μl of the reaction composition solution, and after stirring, 4 μl of each was dispensed into 4 microtubes, and each of the 4 biotinylated dideoxynucleotides (G, A,
2 μl of each of T and C) was added, and a sequence reaction was carried out under the reaction conditions shown below. After stopping the reaction by adding the attached reaction stop solution, heat denaturation at 80 ° C for 2 minutes,
3 μl of each was prepared on a 30 × 40 cm gel plate 5
Apply to 6% acrylamide gel containing 0% urea, 40W
-Electrophoresis was performed for 1.5 hours.

The reaction conditions are as follows: i) heat denaturation: 95 ° C., 30 seconds annealing / extension reaction: 60 ° C., 120 seconds ii) heat denaturation: 95 ° C., 30 seconds annealing: 60 ° C., 30 seconds extension Reaction: 72 ° C., 120 seconds, where i) is cycled 15 times and ii) is cycled 1 time.
It was performed 5 times (30 times in total).

(3) Detection Detection of the sequence was carried out using the reagents attached to the above sequence kit. The method is shown below. After the electrophoresis was completed, one of the gel plates was peeled off, and the DNA in the gel was transferred to the nylon membrane attached to the kit according to the protocol. Then, the biotin-labeled DNA transferred onto the membrane was
Reacted with streptavidin reagent and biotinylated alkaline phosphatase reagent. After washing the membrane with a washing buffer, it is reacted with a luminescent substrate PPD, and the chemiluminescence utilizing the activity of alkaline phosphatase causes the position of the DNA band incorporating the biotinylated dideoxynucleotide separated by electrophoresis to be located on the X-ray film. Detected. After that, the ladder-like band on the X-ray film was read, and the gene sequences of each were compared (FIG. 2).

(4) Results As a result of decoding the band on the X-ray film, a single nucleotide substitution (C → T) not found in the sequence of the wild strain was confirmed in the region involved in the acquisition of rifampicin resistance in the case of sputum 2. Was done.
Due to this mutation, serine at amino acid number 531 of the RNA polymerase β subunit was predicted to be mutated to leucine. This type of mutation has already been reported (Lancet; 341: 647, 1993).
It was speculated that the Mycobacterium tuberculosis infecting this patient is resistant to rifampicin.

When the minimum inhibitory concentration against rifampicin was measured using the isolate actually isolated from the sputum, the value was 128 to 256 ng / ml, and it was confirmed that the strain was obviously resistant.

From this, it was proved that the gene mutation involved in the rifampicin resistance of Mycobacterium tuberculosis existing in the clinical specimen can be analyzed rapidly and with high sensitivity.

[0053]

INDUSTRIAL APPLICABILITY According to the present invention, the RNA polymerase β subunit gene fragment of Mycobacterium tuberculosis can be amplified easily, rapidly and specifically from a clinical sample. This DNA fragment contains a region involved in the acquisition of resistance to rifampicin of Mycobacterium tuberculosis, and the presence or absence of resistance to rifampicin of the bacterium can be estimated by analyzing the mutation in this amplified DNA. An oligonucleotide having a part or all of the sequence of the present invention or its complementary sequence can be used as a primer for a DNA amplification reaction and a sequencing reaction. According to the present invention, the time can be greatly shortened as compared with the conventional drug susceptibility test by culturing, which takes several weeks, and simple and reproducible and reliable results can be obtained.

[0054]

[Sequence list]

[0055]

[SEQ ID NO: 1] Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Determine features Method: S Other characteristics: Mycobacterium tuberculosi
s) It has a sequence complementary to the sequence of RNA polymerase gene.

Sequence GAGGCGATCA CACCGCAGAC GT 22

[0057]

[SEQ ID NO: 2] Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Determine features Method: S Other characteristics: Mycobacterium tuberculosi
s) It has a sequence complementary to the sequence of RNA polymerase gene.

Sequence GATGTTGGGC CCCTCAGGGG TT 22

[Brief description of the drawings]

FIG. 1 is an agarose gel electrophoresis photograph showing the results of Example 2.

FIG. 2 shows comparison of gene sequences of Mycobacterium tuberculosis (wild strain) and each sample of Example 3.

Claims (5)

[Claims] 1. A Mycobacterium Tuberculosis RNA polymerase β containing a nucleic acid sequence consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to said sequence.
Oligonucleotide for subunit gene amplification. 2. The oligonucleotide for amplifying Mycobacterium tuberculosis RNA polymerase β subunit gene according to claim 1, wherein the oligonucleotide is an oligonucleotide modified with a modified product. 3. The oligonucleotide for amplifying Mycobacterium tuberculosis RNA polymerase β subunit gene according to claim 2, wherein the modified product is a radioisotope, a fluorescent substance, a luminescent substance, biotin, and digoxigenin. 4. A method for amplifying a Mycobacterium tuberculosis RNA polymerase β subunit gene using the oligonucleotide according to claim 1 or 2. 5. A DNA obtained by amplifying a nucleic acid in a sample using the oligonucleotide according to claim 1 or 2.
A method of analyzing the presence or absence of base substitution in the DNA using a fragment as a sample.