Oligonucleotide for detection of mycobacterium kansasii group and method for detection thereof

Abstract

To provide oligonucleotide primers and oligonucleotide probes that specifically amplify and detect nucleic acids derived from Mycobacterium kansasii group present in a sample, and a detection method using the same.SOLUTION: The present invention provides a primer set as oligonucleotides for detecting a specific nucleotide sequence or its complementary sequence of DNA or RNA derived from Mycobacterium kansasii group, the primer set comprising at least a combination of a first primer being SEQ ID NO:8 or SEQ ID NO:11 and a second primer being SEQ ID NO:6 or SEQ ID NO:9, and a detection method using the same.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to oligonucleotides for rapidly, highly sensitively and specifically detecting Mycobacterium kansasii group bacteria contained in a sample, and a method for detecting Mycobacterium kansasii group bacteria using the oligonucleotides, and particularly relates to a method for detecting Mycobacterium kansasii group bacteria by an isothermal nucleic acid amplification method.

Background Art

[0002] Mycobacterium kansasii, which is a type of non-tuberculous mycobacteria, is known to be pathogenic to humans. It mainly infects the human lungs and causes lung diseases, and rarely infects lymph nodes, skin, urinary organs, etc. and causes various lesions. Mycobacterium kansasii is an environmental resident bacterium like other non-tuberculous mycobacteria, and environmental water, especially tap water, is considered to be the source of infection. The number of patients with kansasii disease is said to be the second largest among non-tuberculous mycobacteriosis patients in Japan after MAC disease. Also, while the clinical symptoms of pulmonary kansasii disease are known to be almost the same as those of tuberculosis, it is considered that, unlike tuberculosis, it does not infect from human to human. Therefore, it is considered very important to identify the causative bacterium in addition to diagnosis based on clinical symptoms for the negation of tuberculosis and the determination of treatment policies. Furthermore, there is a therapeutic drug that is uniquely effective among non-tuberculous mycobacteria for Mycobacterium kansasii, and it is said to be relatively easy to cure. From this, it is considered very important to simply, rapidly and accurately identify Mycobacterium kansasii for the early determination of treatment policies.

[0003] In general, the identification test for Mycobacterium kansasi is performed by culture methods, nucleic acid amplification methods, etc. The culture method is a method of identifying bacterial species by culturing bacteria from a biological sample and then performing mass spectrometry or DNA-DNA hybridization. It is useful in that it can identify a large number of bacterial species at once, but it takes several weeks to culture the bacteria, so it cannot be diagnosed early, and there is also a problem that the operation is complicated. Nucleic acid amplification methods include the LAMP method (Non-Patent Document 1), the PCR method (Patent Documents 1, 2, 3), etc. Although both sensitivity and specificity are high, the process of extracting nucleic acids derived from bacteria from the sample is complicated, and a certain level of skill of the operator is required to obtain reliable results. In addition, although many nucleic acid amplification techniques have been reported so far, a technique that can amplify nucleic acids isothermally under low-temperature conditions where time is shortened is desired.

[0004] In the TRC method (Patent Documents 4, 5, Non-Patent Document 2) used in the present invention, an integrated device from purification to detection is commercially available. Since the result can be obtained in about 1 hour while maintaining high sensitivity and high specificity, it has high sensitivity, high specificity, and sufficient rapidity.

[0005] More than 180 species of Mycobacterium genus bacteria to which Mycobacterium kansasi belongs have been reported and are very diverse, while many of their base sequences have high homology. Therefore, it has been extremely difficult to design a primer set or oligonucleotide probe that can specifically detect the Mycobacterium kansasi gene and can distinguish only Mycobacterium kansasi. In particular, when using an amplification method that can amplify RNA under relatively low constant temperature conditions (for example, 40°C to 50°C), the amplification target nucleic acid is likely to form a higher-order structure, so the design of the primer set or oligonucleotide probe is even more difficult.

[0006] Furthermore, Mycobacterium kansasii had seven subtypes from subtype 1 to subtype 7. However, these subtypes were reclassified as the Mycobacterium kansasii complex in 2019. Among them, subtype 1 was classified as Mycobacterium kansasii, subtype 2 as Mycobacterium persicum, subtype 3 as Mycobacterim pseudokansasii, subtype 5 as Mycobacterium innocens, and subtype 6 as Mycobacterium attenuatum respectively (Non-Patent Document 3). However, even after reclassification, since their pathogenicity to humans and effective drugs have not changed, it is clinically desirable to be able to widely detect the Mycobacterium kansasii complex in a single measurement.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present invention is to provide a primer set, an oligonucleotide probe for rapidly, highly sensitively and specifically amplifying and detecting nucleic acids derived from Mycobacterium kansasi group present in a sample, and a method for detecting Mycobacterium kansasi group using the same. In particular, it is to provide a method for detecting Mycobacterium kansasi group by an isothermal nucleic acid amplification method.

Effects of the Invention

[0010] The method for detecting a specific base sequence of Mycobacterium kansasi group of the present invention provides a primer set consisting of a first primer and a second primer for amplifying the specific base sequence, a nucleic acid amplification method, and an oligonucleotide probe for nucleic acid detection, thereby providing a method for rapidly, highly sensitively and specifically detecting Mycobacterium kansasi group.

Means for Solving the Problems

[0011] As a result of intensive studies to solve the above problems, the present inventor has completed the present invention. That is, the present invention is as follows. (1) A primer set for detecting Mycobacterium kansasi group, the primer set comprising at least a first primer and a second primer, and the combination of the first primer and the second primer is selected from the base sequences described in any of the following (A) to (D) or their complementary strands. (A) First primer: SEQ ID NO: 8, second primer: SEQ ID NO: 6 (B) First primer: SEQ ID NO: 8, second primer: SEQ ID NO: 9 (C) First primer: SEQ ID NO: 11, second primer: SEQ ID NO: 6 (D) First primer: SEQ ID NO: 11, second primer: SEQ ID NO: 9 The primer set according to (1), wherein the first primer is SEQ ID NO: 8. The primer set according to (1), further comprising the oligonucleotide probe according to SEQ ID NO: 12, wherein the oligonucleotide is labeled with a fluorescent dye and is configured such that the fluorescence characteristics change when complementary double strands are formed. (4) The primer set according to (1), wherein the first primer and the second primer each have a transcribable promoter sequence of RNA polymerase added to at least one 5'-end. (5) The primer set according to (4), wherein the promoter sequence is SEQ ID NO: 13. (6) A step of synthesizing cDNA complementary to a specific base sequence using a second primer and an enzyme having RNA-dependent DNA polymerase activity, with the specific base sequence of the Mycobacterium kansasi group as a template; [2] A step of decomposing the RNA of the RNA-DNA double strand by an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA); [3] A step of generating double-stranded DNA having a promoter sequence capable of transcribing RNA of a specific base sequence or a sequence complementary to the specific base sequence using the single-stranded DNA as a template by a first primer and an enzyme having DNA-dependent DNA polymerase activity; [4] A step of generating an RNA transcription product using the double-stranded DNA in the above step as a template by an enzyme having RNA polymerase activity; [5] A step of continuously generating an RNA transcription product by using the RNA transcription product as a template for cDNA synthesis in the step [1], and [6] A method for specifically detecting the Mycobacterium kansasi group, including a step of detecting the generated RNA transcription product using an oligonucleotide probe whose fluorescence characteristics change when forming a double strand with a partial base sequence of the transcription product as compared with before formation, and using any of the primer sets of (1) to (5) in the above method. (7) The method further includes a step of cutting a region overlapping with the 5'-terminal side of the homologous region of the template RNA in [1] with the first primer of the specific base sequence and adjacent to the 5'-terminal side from the overlapping site with a cleavage oligonucleotide. (8) For the cleavage oligonucleotide, when the first primer is SEQ ID NO: 8, it is an oligonucleotide consisting of the base sequence of SEQ ID NO: 7, and when the first primer is SEQ ID NO: 11, it is an oligonucleotide consisting of the base sequence of SEQ ID NO: 10, according to the method described in (7) above. (9) The method according to (6) above, wherein the enzyme is selected from the group consisting of AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molony Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, and HIV (Human Immunodeficiency Virus) reverse transcriptase. (10) The method according to (9) above, wherein the enzyme is AMV reverse transcriptase. (11) The method according to (10) above, wherein the steps [1] to [6] are carried out within a temperature range of 30 to 65 °C. (12) The method according to (11) above, wherein the steps [1] to [6] are carried out within a temperature range of 40 to 50 °C. (13) The method according to (6) above, wherein the enzyme having RNA polymerase activity used in the step [4] is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. (14) The method according to (13) above, wherein the enzyme having RNA polymerase activity is T7 RNA polymerase.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The present invention will be described in detail below.

[0013] The present invention relates to an oligonucleotide primer and an oligonucleotide probe for specifically detecting the Mycobacterium kansasii complex present in a sample, and a detection method using the oligonucleotide primer and the oligonucleotide probe.

[0014] In the present invention, the sample includes body fluids such as sputum, gastric juice, pleural effusion, ascites, urine, biological samples such as bronchial lavage fluid, tissue, culture fluid, or DNA or RNA extracted from the biological sample.

[0015] In the present invention, the Mycobacterium kansasii complex is the old classification of Mycobacterim kansasii subtype 1 to subtype 7, that is, it includes Mycobacterium persicum, Mycobacterium pseudokansasii, Mycobacterium innocens, and Mycobacterium attenuatum described above.

[0016] The specific nucleotide sequence in the present invention refers to DNA or RNA consisting of a continuous nucleotide sequence of 250 nucleotides or less or its complementary sequence, which contains the nucleotide sequences described in SEQ ID NO: 1 (nucleotide sequence from position 1416181 to position 1416439 of GenBank No. CP006835.1) and SEQ ID NO: 2 (nucleotide sequence from position 26670 to position 26926 of GenBank No. MVIF01000072.1) among the gene sequences of the Mycobacterium kansasi group.

[0017] The first primer and the second primer in the present invention are oligonucleotides consisting of a continuous sequence of 15 nucleotides or more selected from the nucleotide sequences described in SEQ ID NO: 1 or SEQ ID NO: 2 or their complementary sequences.

[0018] Preferred embodiments of the first primer and the second primer in the present invention include oligonucleotides consisting of the nucleotide sequences described in any of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or their complementary strands. For example, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11 can be used as the first primer, and SEQ ID NO: 6, SEQ ID NO: 9 can be used as the second primer. More preferably, SEQ ID NO: 8, SEQ ID NO: 11 can be used as the first primer, and SEQ ID NO: 6, SEQ ID NO: 9 can be used as the second primer. These oligonucleotides are applicable even if base mutations, deletions, additions, or chemical modifications are made within the range where their specificity does not change in hybridization with the target nucleic acid.

[0019] The oligonucleotide probe in the present invention is for detecting the presence of DNA or RNA of the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or its complementary sequence, and is an oligonucleotide consisting of at least a continuous 15 nucleotides or its complementary strand among the nucleotide sequences described in SEQ ID NO: 1 or SEQ ID NO: 2.

[0020] As a preferred embodiment of the oligonucleotide probe in the present invention, there is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 12 or its complementary strand. Note that, as in the case of primers, it is applicable even if base mutations, deletions, additions, or chemical modifications are made as long as there is no change in its specificity in hybridization with the target nucleic acid.

[0021] For the detection of the specific base sequence of the Mycobacterium kansasi group or its complementary sequence using the oligonucleotide probe of the present invention, conventionally known nucleic acid detection methods can be utilized. Specifically, there are (A) methods using electrophoresis or liquid chromatography, (B) hybridization methods using oligonucleotide probes labeled with detectable labeling substances, (C) methods using a fluorescent dye-labeled oligonucleotide probe designed such that the fluorescence characteristics change by hybridizing with a part of the base sequence of an amplification product obtained by amplifying a nucleic acid containing the specific base sequence or its complementary sequence using a set of oligonucleotide primers, and the like. As the labeling substance in the above (B), known ones such as enzymes, fluorescent dyes, radioisotopes, and luminescent dyes can be used. Further, as an example of the fluorescent dye-labeled oligonucleotide probe in the above (C), there are fluorescent-labeled probes using FRET (fluorescence resonance energy transfer) and oligonucleotide probes labeled with intercalator fluorescent dyes. Specifically, when applied to molecular beacon (U.S. Patent No. 5,925,517, U.S. Patent No. 6,103,476), TaqMan probe (U.S. Patent No. 5,210,015, U.S. Patent No. 5,487,972), Q-Probe (Patent No. 3,437,816), cycling probe (U.S. Patent No. 5,011,769, U.S. Patent No. 5,403,711), intercalator fluorescent dye-labeled probe (INAF probe, Patent Document 4, Patent Document 5, Non-Patent Document 2, and JP-A 2000-316587), etc., it is preferable because the detection operation can be performed easily.

[0022] Among them, an oligonucleotide probe labeled with an intercalator fluorescent dye and designed such that when it forms a complementary double strand with a target nucleic acid, the fluorescent dye portion intercalates into the complementary double strand portion, thereby changing the fluorescence characteristics, can detect the nucleic acid amplified simultaneously with the nucleic acid amplification reaction described later. Just by adding it to a container together with reagents such as the amplification primer set, enzyme, and enzyme substrate described later, the amplification step and the detection step can be carried out. Therefore, it is particularly preferable as the oligonucleotide primer of the present invention. As an example of the oligonucleotide probe labeled with the intercalator fluorescent dye, a 3'-end, 5'-end, phosphodiester site, or base site of an oligonucleotide complementary to a part of a nucleic acid containing a specific base sequence of the Mycobacterium kansasi group or a complementary sequence of the specific base sequence, with an intercalator fluorescent dye labeled via an appropriate linker, can be mentioned. The intercalator dye to be labeled is not particularly limited, and examples include cyanine dyes such as oxazole yellow and thiazole orange, hemicyanine dyes, azo dyes such as ethidium bromide and methyl red, or derivatives thereof, but it is not limited to this. In addition, when detection is also carried out during the nucleic acid amplification step, for the purpose of preventing the nucleic acid elongation reaction, it is preferable that the hydroxyl group on the 3'-end side of the oligonucleotide probe is modified with a phosphate or glycolic acid or the like.

[0023] Normally, the abundance of nucleic acid derived from the Mycobacterium kansasi group present in a clinically obtained sample is trace. Therefore, when detecting Mycobacterium kansasi present in a sample using the oligonucleotide probe of the present invention, it is advisable to amplify the nucleic acid derived from Mycobacterium kansasi present in the sample by a nucleic acid amplification method or to detect it simultaneously with amplification. The nucleic acid amplification method is not particularly limited, and known methods can be used. Examples include the PCR method (Patent Documents 1 to 3), RT-PCR method, LAMP method (Non-Patent Document 1), TRC method (Patent Documents 4, 5, and Non-Patent Document 2), NASBA method (Japanese Patent No. 2650159), and TMA method (Japanese Patent No. 3241717), but it is not limited to this.

[0024] The primer set in the present invention includes at least a first primer and a second primer, and is useful as a primer for amplifying a nucleic acid containing a specific base sequence derived from Mycobacterium kansasi or a complementary sequence of the specific base sequence using the amplification method. When a promoter of RNA polymerase is further added to the 5'-end side of either the first primer or the second primer, a nucleic acid containing a specific base sequence to which the promoter of RNA polymerase is added or a complementary sequence of the specific base sequence is synthesized using the RNA polymerase corresponding to the promoter. Therefore, it is preferable in that RNA can be amplified using a method for amplifying RNA at a constant temperature such as the NASBA method, the TMA method, or the TRC method from these nucleic acids. As the promoter to be added to the 5'-end side of the primer, a promoter corresponding to the RNA polymerase used for RNA amplification (for example, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase widely used in the field of molecular biology) may be used. Further, a transcription start region known to affect transcription efficiency may be further added to the promoter. As a specific example of the promoter (T7 promoter) added to the 5'-end side of the primer when T7 RNA polymerase is used as the RNA polymerase used for RNA amplification, an oligonucleotide having the sequence described in SEQ ID NO: 13 can be mentioned.

[0025] To amplify and detect the Mycobacterium kansasi group using the oligonucleotide probe and primer set in the present invention, for example, it may be carried out by the steps shown in the following (1) to (6). (1) A step of synthesizing cDNA complementary to a specific base sequence using RNA derived from Mycobacterium kansasi as a template with a second primer and an enzyme having RNA-dependent DNA polymerase activity. (2) A step of decomposing the RNA of the RNA-DNA double strand with an enzyme having ribonuclease H (RNaseH) activity (generation of single-stranded DNA), (3) Generating double-stranded DNA having a specific base sequence using the single-stranded DNA as a template by a first primer and an enzyme having DNA-dependent DNA polymerase activity, or having a promoter sequence capable of transcribing RNA of a sequence complementary to the specific base sequence. (4) Producing an RNA transcription product using the double-stranded DNA as a template by an enzyme having RNA polymerase activity. (5) Sequentially producing an RNA transcription product by using the RNA transcription product as a template for cDNA synthesis in the reaction of (1). (6) Detecting the amount of the RNA transcription product over time using an oligonucleotide probe labeled with an intercalating fluorescent dye on an oligonucleotide capable of hybridizing to the RNA transcription product. The enzyme having RNA-dependent DNA polymerase activity used in the step (1), the enzyme having RNaseH activity used in the step (2), and the enzyme having DNA-dependent DNA polymerase activity used in the step (3) can be added individually or in various combinations, but a reverse transcriptase derived from a retrovirus having the above activities can also be used. The reverse transcriptase is not particularly limited, but AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molony Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, HIV (Human Immunodeficiency Virus) reverse transcriptase, etc., which are widely used in the field of molecular biology, can be used.

[0026] In order to carry out the amplification reaction described above, in addition to the sample and each of the enzymes, a buffer, a magnesium salt, a potassium salt, deoxynucleoside triphosphate, ribonucleoside triphosphate, etc. are added. Further, if necessary to adjust the reaction efficiency, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), bovine serum albumin (BSA), sugars such as trehalose, etc. may be added. Also, for the temperature conditions of the amplification reaction, a reaction temperature suitable for the enzyme used may be set. For example, when the enzymes used in the above step are AMV reverse transcriptase and T7 RNA polymerase, the reaction temperature may be set in the range of 35°C to 65°C, and more preferably in the range of 40°C to 50°C.

[0027] In the above-mentioned step, a promoter sequence may be added to the 5'-end of either the first primer or the second primer. However, when adding a promoter sequence to the first primer, before carrying out the step (1) above, or simultaneously with the step (1) above, it is preferable that there is a region that overlaps with the 5'-end side of the homologous region of the first primer of a specific base sequence in the template RNA and is adjacent to the 5'-end side from the overlapping site and has been previously cleaved. By cleaving the specific base sequence, after cDNA synthesis, a DNA strand complementary to the promoter sequence of the first primer hybridized to the cDNA can be efficiently synthesized by extending the 3'-end of the cDNA, and as a result, a double-stranded DNA containing a promoter capable of transcribing RNA can be generated. As a preferable example of the cleavage method, an oligonucleotide having a sequence complementary to a part of the specific base sequence of RNA derived from the Mycobacterium kansasi group, and more preferably an oligonucleotide having a complementary sequence that overlaps or is adjacent to the 5'-side of the homologous region of the first primer of the specific base sequence (hereinafter referred to as the cleavage oligonucleotide) is added, and then the RNA portion of the RNA-DNA hybrid formed thereby is cleaved by an enzyme having RNase H activity, etc. It should be noted that the hydroxyl group at the 3'-end of the cleavage oligonucleotide is preferably appropriately modified, such as with phosphate, etc., in order to prevent the extension reaction. As an example of the cleavage oligonucleotide used for amplifying RNA derived from Mycobacterium kansasi, when the first primer is SEQ ID NO: 4 and SEQ ID NO: 5, it is the oligonucleotide consisting of the base sequence described in SEQ ID NO: 3; when the first primer is SEQ ID NO: 8, it is the oligonucleotide consisting of the base sequence described in SEQ ID NO: 7; when the first primer is SEQ ID NO: 11, it is the oligonucleotide consisting of the base sequence described in SEQ ID NO: 10.

[0028] The method for detecting Mycobacterium kansasi flora of the present invention can detect rapidly and with high sensitivity while targeting one gene region of the Mycobacterium kansasi flora contained in a sample, and has extremely low false positive occurrence and high specificity, such as not detecting other Mycobacterium spp. Therefore, it becomes possible to promptly present the test result to a doctor, which is effective for preventing the spread of infection and determining an appropriate treatment policy, and is considered to contribute to the early improvement of symptoms and public health.

[0029] The primer set and oligonucleotide probe of the present invention, and the detection method using the primer set and the oligonucleotide probe can rapidly, highly sensitively and specifically detect Mycobacterium kansasi flora by providing a primer set consisting of a first primer and a second primer for amplifying a specific base sequence derived from Mycobacterium kansasi, an oligonucleotide probe for nucleic acid detection, and a nucleic acid amplification method.

[0030] The primer set and oligonucleotide probe of the present invention, and the method for detecting Mycobacterium kansasi flora using the oligonucleotide can rapidly and highly sensitively detect Mycobacterium abscessus complex bacteria contained in a sample and have high specificity. Therefore, it becomes possible to promptly present the test result to a doctor, which is effective for determining an appropriate treatment policy, and is considered to contribute to the early improvement of symptoms and public health.

Example

[0031] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[0032] Example 1 Preparation of synthetic RNA or diluted bacterial culture solution The synthetic RNA of Mycobacterium spp. used in the examples described below was prepared by the following method. The quantification of the prepared synthetic RNA was carried out based on the absorbance at 260 nm. (1) Mycobacterium kansasii synthetic RNA An artificial 16S rRNA gene was prepared based on the 16S rRNA sequence of Mycobacterium kansasii (Mycobacterium kansasii subtype 1), in vitro transcribed, and then the transcription product was purified to prepare Mycobacterium kansasii synthetic RNA. (2) Mycobacterium persicum synthetic RNA An artificial 16S rRNA gene was prepared based on the 16S rRNA sequence of Mycobacterium persicum (Mycobacterium kansasii subtype 2), in vitro transcribed, and then the transcription product was purified to prepare Mycobacterium persicum synthetic RNA. (3) Mycobacterium tuberculosis synthetic RNA A 16S rRNA gene was created based on the 16S rRNA sequence of Mycobacterium tuberculosis, in vitro transcribed, and then the transcription product was purified to prepare Mycobacterium tuberculosis synthetic RNA. (4) Mycobacterium avium synthetic RNA A 16S rRNA gene was created based on the 16S rRNA sequence of Mycobacterium avium, in vitro transcribed, and then the transcription product was purified to prepare Mycobacterium avium synthetic RNA. (5) Mycobacterium intracellulare synthetic RNA Based on the 16S rRNA sequence of Mycobacterium intracellulare, a 16S rRNA gene was prepared, in vitro transcribed, and then the transcription product was purified to prepare Mycobacterium intracellulare synthetic RNA.

[0033] Example 2 Preparation of Oligonucleotide Labeled with Intercalator Fluorescent Dye An oligonucleotide labeled with an intercalator fluorescent dye (hereinafter referred to as an oligonucleotide probe) shown in the following (A) was prepared based on the method disclosed in JP-A-2000-316587. (A) An oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 12 or its complementary sequence, wherein in SEQ ID NO: 12, thiazole orange is labeled via a linker between the 8th guanosine and the 9th cytidine from the 5'-end.

[0034] Example 3 Examination of Oligonucleotide for Detecting Mycobacterium kansasi Group Using the combination of the first primer and the second primer (hereinafter referred to as the combination of oligonucleotides) shown in Table 1, evaluation was performed by the method shown below. The oligonucleotide probe described in Table 1 is the probe prepared in Example 2. (1) Among the synthetic RNAs prepared in Example 1, Mycobacterium kansasi synthetic RNA (Example 1(1)) and Mycobacterium persicum synthetic RNA (Example 1(2)) were diluted to 1000 copies / 15 μL using an RNA dilution solution (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.02% sodium cholate), and these were used as RNA samples. (2) A reaction solution having the following composition was dispensed into an evaporation drying tube and evaporated to dryness.

[0035] Composition of reaction solution: Concentration is RNA sample, starting solution, final concentration (in 30 μL) after addition 60 mM Tris-HCl buffer (pH 8.35) 150 mM trehalose 0.30 mM each of dATP, dCTP, dGTP, dTTP 3.0 mM each of ATP, CTP, UTP 2.1 mM GTP 3.4 mM ITP 0.16 μM oligonucleotide for cleavage 0.5 μM first primer (with a T7 promoter (SEQ ID NO: 13) added to the 5'-end of an oligonucleotide consisting of the nucleotide sequence of each SEQ ID NO) 0.5 μM second primer 35 nM oligonucleotide probe (prepared in Example 2) 0.025 mg / mL bovine serum albumin 150 U T7 RNA polymerase 6.4 U AMV reverse transcriptase (3) After adding 15 μL of the RNA sample after the above evaporation to dryness, incubate at 46 °C for 5 minutes, and then add 15 μL of the starting solution consisting of the following composition and stir.

[0036] Composition of the enzyme solution: final concentration during the reaction (in 30 μL) 11% dimethyl sulfoxide 21 mM magnesium chloride 85 mM potassium chloride (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function that allows direct measurement of the evaporation tube, react at 46 °C and simultaneously measure the fluorescence intensity of the reaction solution over time for 20 minutes.

[0037] Taking the time point when the addition of the starting solution and the stirring were completed as 0 minutes, when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.20, it was judged as positive, and the time at that moment was taken as the detection time. The results are shown in Table 1. "N.D." means that the fluorescence intensity ratio after 20 minutes from the start of the reaction was less than 1.20 (negative judgment).

[0038] The results are shown in Table 1. The oligonucleotide combinations C2 described in JP-A-2004-194583, and the oligonucleotide combinations C1, C3, and C4 were unable to detect Mycobacterium kansasi group bacteria, whereas the oligonucleotide combinations C5, C6, C7, and C8 were able to detect Mycobacterium kansasi bacteria within 10 minutes. Thus, it can be said that these are oligonucleotide combinations capable of rapidly detecting Mycobacterium kansasi bacteria.

[0039] [Table 1]

[0040] Example 4 Specificity of Oligonucleotides for Detecting Mycobacterium kansasi Group Bacteria Using the oligonucleotide combinations C5, C6, C7, and C8 described in Table 1, the evaluation was performed by the method shown below. The oligonucleotide probes described in Table 1 are the probes prepared in Example 2. (1) Among the standard RNAs prepared in Example 1, Mycobacterium tuberculosis synthetic RNA (Example 1(3)), Mycobacterium avium synthetic RNA (Example 1(4)), and Mycobacterium intracellulare synthetic RNA (Example 1(5)) were diluted to 100,000 copies / 15 μL using an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.02% sodium cholate), and these were used as RNA samples. (2) A reaction solution having the following composition was dispensed into a tube for evaporation to dryness and evaporated to dryness.

[0041] Composition of reaction solution: Concentrations are for RNA sample, starting solution, and final concentration in (30 μL) after addition 60 mM Tris-HCl buffer (pH 8.35) 150 mM trehalose 0.30 mM each of dATP, dCTP, dGTP, dTTP 3.0 mM each of ATP, CTP, UTP 2.1 mM GTP 3.4 mM ITP 0.16 μM Oligonucleotide for cleavage 0.5 μM First primer (T7 promoter (SEQ ID NO: 13) added to the 5'-end of the oligonucleotide consisting of the nucleotide sequence of each SEQ ID NO) 0.5 μM Second primer 35 nM Oligonucleotide probe (prepared in Example 2) 0.025 mg / mL Bovine serum albumin 150 U T7 RNA polymerase 6.4 U AMV reverse transcriptase (3) After adding 15 μL of the RNA sample after the above evaporation to dryness, incubate at 46 °C for 5 minutes, and then add 15 μL of the starting solution having the following composition and stir.

[0042] Composition of enzyme solution: Final concentration during reaction (in 30 μL) 11% Dimethyl sulfoxide 21 mM Magnesium chloride 85 mM Potassium chloride (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of direct measurement of the evaporation-to-dryness tube, react at 46 °C and simultaneously measure the fluorescence intensity of the reaction solution over time for 20 minutes.

[0043] When the time point when the addition of the starting solution and the stirring were completed was taken as 0 minute, when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.20, it was judged as positive, and the time at that time was taken as the detection time. The results are shown in Table 1. "N.D." means that the fluorescence intensity ratio after 20 minutes from the start of the reaction was less than 1.20 (negative judgment). The results are shown in Table 2. Since all of the combinations C5, C6, C7, and C8 of the oligonucleotides were negative judgments (N.D.), it can be said that these combinations of oligonucleotides can specifically detect Mycobacterium kansasiibacteria.

[0044]

Table 2

Claims

1. A primer set for detecting Mycobacterium kansasi group, the primer set comprising at least a first primer and a second primer, and the combination of the first primer and the second primer being selected from the nucleotide sequences described in any of the following (A) to (D) or their complementary strands. (A) First primer: SEQ ID NO: 8, Second primer: SEQ ID NO: 6 (B) First primer: SEQ ID NO: 8, Second primer: SEQ ID NO: 9 (C) First primer: SEQ ID NO: 11, Second primer: SEQ ID NO: 6 (D) First primer: SEQ ID NO: 11, Second primer: SEQ ID NO: 9

2. The primer set according to claim 1, wherein the first primer is SEQ ID NO:

8.

3. The primer set according to claim 1, further comprising an oligonucleotide probe described in SEQ ID NO: 12, which is an oligonucleotide labeled with a fluorescent dye and configured such that the fluorescence characteristics change when complementary double-strands are formed.

4. The primer set according to claim 1, wherein a transcribable promoter sequence of RNA polymerase is added to at least one 5'-end of the first primer and the second primer.

5. The primer set according to claim 4, wherein the promoter sequence is SEQ ID NO:

13.

6. (1) A step of synthesizing cDNA complementary to a specific nucleotide sequence using a specific nucleotide sequence of Mycobacterium kansasi group as a template by a second primer and an enzyme having RNA-dependent DNA polymerase activity; (2) A step of degrading RNA of the RNA-DNA double-strand (generation of single-stranded DNA) by an enzyme having ribonuclease H (RNase H) activity; (3) A step of generating double-stranded DNA having a transcribable promoter sequence of a specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence using the single-stranded DNA as a template by a first primer and an enzyme having DNA-dependent DNA polymerase activity; (4) A step of generating an RNA transcription product using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; (5) A step of continuously generating an RNA transcription product by using the RNA transcription product as a template for cDNA synthesis in the step (1). (6) A method for specifically detecting Mycobacterium kansasi group bacteria, comprising the step of detecting the generated RNA transcript using an oligonucleotide probe whose fluorescence characteristics change when forming a double strand with a partial base sequence of the transcript as compared with before the formation, wherein the method uses any of the primer sets according to claims 1 to 5.

7. The method according to claim 6, further comprising a step of overlapping with the 5'-end side of the homologous region with the first primer of a specific base sequence among the template RNAs of (1) and being cut with a cleavage oligonucleotide in a region adjacent to the 5'-end side from the overlapping site.

8. Regarding the cleavage oligonucleotide, when the first primer is SEQ ID NO: 8, it is an oligonucleotide consisting of the base sequence of SEQ ID NO: 7, The method according to claim 7, which is an oligonucleotide consisting of a base sequence that is SEQ ID NO: 10 when the first primer is SEQ ID NO:

11.

9. The method according to claim 6, wherein the enzyme is selected from the group consisting of AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Moloney Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, and HIV (Human Immunodeficiency Virus) reverse transcriptase.

10. The method according to claim 9, wherein the enzyme is AMV reverse transcriptase.

11. The method according to claim 10, wherein the steps (1) to (6) are carried out within a temperature range of 30 to 65°C.

12. The method according to claim 11, wherein the steps (1) to (6) are carried out within a temperature range of 40 to 50°C.

13. The method according to claim 6, wherein the enzyme having RNA polymerase activity used in the step (4) is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

14. The method according to claim 13, wherein the enzyme having RNA polymerase activity is T7 RNA polymerase.

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