Oligonucleotides used for detecting Mycobacterium abscesses complex and methods for detecting the same

Abstract

To provide a detection method that specifically amplifies Mycobacterium abscessus complex bacteria in a sample and can reduce a false-positive rate.SOLUTION: The present invention provides an oligonucleotide probe for detecting a specific base sequence of 23S rRNA or 23S rDNA of Mycobacterium abscessus complex bacteria or its complementary sequence, the oligonucleotide consisting of at least continuous 14 bases of a base sequence described in SEQ ID NO: 3 or a complementary sequence of the sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an oligonucleotide for rapidly, highly sensitively, and specifically detecting Mycobacteroides abscessus complex bacteria contained in a sample, and a method for detecting Mycobacteroides abscessus complex bacteria using the oligonucleotide.

Background Art

[0002] Mycobacteroides abscessus complex, which is a type of non-tuberculous mycobacteria, is the causative bacterium of abscessus disease and is pathogenic to humans. Mycobacteroides abscessus complex 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 abscessus disease is the third highest among non-tuberculous mycobacteriosis patients in Japan, after MAC disease and kansashi disease, and it is said that abscessus disease has been increasing in recent years. The infection site in abscessus disease is mainly the lungs, causing lung diseases, and rarely infects lymph nodes, skin, urinary organs, etc., causing various lesions. Also, while the clinical symptoms of pulmonary abscessus disease are known to be almost the same as those of tuberculosis, it is considered that, unlike tuberculosis, human-to-human infection does not occur. Therefore, in addition to diagnosis based on clinical symptoms, it is very important to identify the causative bacterium for the negation of tuberculosis and the determination of treatment policies. From this, it is very important to simply, rapidly, and accurately identify Mycobacteroides abscessus complex for the early formulation of treatment policies.

[0003] Generally, Mycobacterium abscesses complex is identified using methods such as DNA-DNA hybridization (Patent Document 1) and mass spectrometry. While DNA-DNA hybridization and mass spectrometry are useful because they can identify multiple bacterial species at once, they lack speed because they require bacterial culture using the sample, making early diagnosis difficult. Therefore, there is a need for a highly sensitive and simple test that can be performed quickly without the need for culture.

[0004] The TRC method used in this invention (Patent Documents 2 and 3) utilizes a commercially available integrated apparatus that handles everything from purification to detection, and results can be obtained in about an hour, thus offering sufficient speed. Furthermore, its sensitivity and specificity are comparable to other nucleic acid amplification detection methods.

[0005] The Mycobacterium abscessus complex, to which the non-tuberculous mycobacteria belong, currently comprises over 190 species, exhibiting great diversity, yet many of these species share similar gene sequences. Consequently, designing primer sets and oligonucleotide probes that can distinguish only the Mycobacterium abscessus complex gene with high sensitivity and specificity has been extremely difficult. This is especially true when using amplification methods that allow RNA amplification under relatively low, constant temperature conditions (e.g., 40°C to 50°C), as the target nucleic acid tends to form higher-order structures, making the design of such primer sets and oligonucleotide probes even more challenging. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2006-61155 [Patent Document 2] Japanese Patent Publication No. 2000-14400 [Patent Document 3] Japanese Patent Publication No. 2001-37500 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a specific oligonucleotide that can rapidly and sensitively amplify nucleic acids derived from Mycobacterium abscesses complex bacteria present in a sample, and that is less prone to false positives, and a method for detecting Mycobacterium abscesses complex bacteria using the oligonucleotide. [Means for solving the problem]

[0008] The inventors of this invention have diligently conducted research to solve the above problems and have now completed this invention.

[0009] In other words, the present invention is as follows. (1) An oligonucleotide for detecting a specific nucleotide sequence or complementary sequence of the 23S rRNA or 23S rDNA of Mycobacterium abscesses complex, characterized in that it consists of at least 14 consecutive nucleotides of the nucleotide sequence described in Sequence ID No. 3 or the complementary sequence of said sequence. (2) The oligonucleotide according to (1), characterized in that the oligonucleotide is labeled with a fluorescent dye and configured to change its fluorescence properties when it forms complementary double helix. (3) The oligonucleotide according to (2), characterized in that the oligonucleotide is labeled with an intercalator fluorescent dye. (4) A method for detecting Mycobacterium abscesses complex bacteria, characterized by using an oligonucleotide described in any of (1) to (3) for detecting a specific base sequence or complementary sequence of 23S rRNA or 23S rDNA of Mycobacterium abscesses complex bacteria. (5) A method for detecting Mycobacterium abscesses complex bacteria according to (4), characterized by comprising the step of amplifying a specific base sequence or its complementary sequence using a set of primers. (6) The method for detecting Mycobacterium abscesses complex bacteria according to (4) or (5), characterized in that the set of primers comprises a first primer that hybridizes to the sequence described in Sequence ID No. 9, and / or a second primer that hybridizes to the sequence described in Sequence ID No. 24. (7) A method for detecting Mycobacterium abscesses complex bacteria according to any one of (4) to (6), characterized in that the first primer consists of 19 to 26 consecutive bases in the sequence or complementary sequence described in Sequence ID No. 9, and the second primer consists of 16 to 26 consecutive bases in the sequence or complementary sequence described in Sequence ID No. 24. (8) A reagent kit for detecting Mycobacterium abscesses complex, comprising an oligonucleotide as described in any of (1) to (3) and a primer as described in (6) or (7). (9) The kit according to (8), comprising a first primer having the sequence described in Sequence ID No. 12 and a second primer having the sequence described in Sequence ID No. 25.

[0010] The present invention will be described in detail below.

[0011] In this invention, the term "sample" refers to bodily fluids such as sputum, gastric juice, pleural fluid, ascites, and urine, as well as bronchial lavage fluid, tissue, and culture medium.

[0012] In the present invention, the specific nucleotide sequence of Mycobacterium abscessus complex 23S rRNA or 23S rDNA refers to a sequence of 250 nucleotides or less that includes the nucleotide sequence described in Sequence ID No. 1 (the nucleotide sequence from position 1465775 to position 1466012 of GenBank No. CU458896.1) within the sequence of Mycobacterium abscessus complex 23S rRNA or 23S rDNA of Mycobacterium abscessus complex.

[0013] In other words, in the present invention, nucleic acids containing the specific base sequence or a complementary sequence of the specific base sequence will be detected.

[0014] The detection of nucleic acids containing a specific nucleotide sequence or its complementary sequence of Mycobacterium abscessus complex 23S rRNA using the oligonucleotide of the present invention can be performed using conventionally known nucleic acid detection methods. Specifically, (A) Methods using electrophoresis or liquid chromatography, (B) Hybridization method using oligonucleotide probes labeled with detectable labels, (C) A method using a fluorescent dye-labeled oligonucleotide designed so that its fluorescence properties change when a nucleic acid containing the specific base sequence or its complementary sequence is hybridized with a portion of the base sequence of an amplified product using a set of primers. Examples include fluorescently labeled oligonucleotides of the above (C), such as fluorescently labeled oligonucleotides using FRET (fluorescence resonance energy transfer) and oligonucleotides labeled with intercalator fluorescent dyes.

[0015] An example of an oligonucleotide labeled with the aforementioned intercalator fluorescent dye is an oligonucleotide in which an intercalator fluorescent dye is labeled via an appropriate linker at the 3' end, 5' end, phosphate diester portion, or base portion of an oligonucleotide complementary to a specific nucleotide sequence of Mycobacterium abscessus complex 23S rRNA or a portion of the nucleic acid containing the complementary sequence of said specific nucleotide sequence. When the oligonucleotide forms a complementary double helix with the specific nucleotide sequence of Mycobacterium abscessus complex 23S rRNA (or the complementary sequence of said specific nucleotide sequence), the intercalator fluorescent dye portion intercalates into the complementary double helix, thereby changing its fluorescence properties. There are no particular limitations on the intercalator fluorescent dye used for labeling; commonly used fluorescent dyes such as oxazole yellow, thiazole orange, ethidium bromide, hemicyanin, and their derivatives can be appropriately selected considering fluorescence intensity and fluorescence properties. Except when labeling the 3' end with a fluorescent dye, it is preferable that the 3' end of the oligonucleotide be appropriately modified with glycolic acid or similar to prevent nucleic acid elongation from that end.

[0016] Oligonucleotides constituting the oligonucleotides labeled with the intercalator fluorescent dye include oligonucleotides having a sequence complementary to a nucleic acid containing a specific base sequence or a complementary sequence of the specific base sequence, and containing a continuous base sequence of at least 14 bases or a complementary sequence thereof in the base sequence described in Sequence ID No. 3 (base sequence from 1465926 to 1465942 of GenBank No. CU458896.1). More preferably, oligonucleotides having a sequence complementary to the base sequence or complementary sequence described in Sequence ID No. 5, and oligonucleotides that can hybridize with the base sequence or complementary sequence described in Sequence ID No. 7.

[0017] Examples of conditions in the present invention include conditions where 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate are present at 42°C, and nucleic acid amplification conditions described in the examples of this specification. Further, under the above-described conditions, if the first and second primer base sequences can hybridize specifically and efficiently with the specific base sequence, they may have substitutions, deletions, additions, or modifications compared to the specific base sequence. The lengths of the first and second primers can be arbitrarily set, but are preferably in the range of 10 bases to 50 bases. In the most preferred embodiment, an oligonucleotide capable of specifically hybridizing is an oligonucleotide having a sequence complementary to the target base sequence.

[0018] The present invention uses, as a first primer having a sequence complementary to the 3'-terminal portion of the specific base sequence of Mycobacterium abscessus complex bacterium 23S rRNA contained in a sample, the base sequence described in SEQ ID NO: 10 (the base sequence from position 1465969 to position 1466010 of GenBank No. CU458896.1) or an oligonucleotide capable of specifically hybridizing with its complementary sequence, and also uses, as a second primer having a sequence homologous to the 5'-terminal portion of the specific base sequence of Mycobacterium abscessus complex bacterium 23S rRNA contained in the sample, the base sequence described in SEQ ID NO: 23 (the base sequence from position 1465775 to position 1465918 of GenBank No. CU458896.1) or an oligonucleotide capable of specifically hybridizing with its complementary sequence.

[0019] Among them, it is preferable that the first primer and the second primer each consist of 16 to 26 bases.

[0020] As an example of the first primer, an oligonucleotide consisting of 19 to 26 consecutive bases in the base sequence described in SEQ ID NO: 10 or its complementary sequence can be mentioned. As a more specific example, oligonucleotides consisting of the base sequences described in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 can be mentioned.

[0021] As an example of the second primer, an oligonucleotide consisting of 16 to 26 consecutive bases in the base sequence described in SEQ ID NO: 23 or its complementary sequence can be mentioned. As a more specific example, oligonucleotides consisting of the base sequences described in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 can be mentioned.

[0022] In particular, a primer set is preferred in which the first primer is an oligonucleotide consisting of any of the nucleotide sequences described in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, and the second primer is an oligonucleotide consisting of any of the nucleotide sequences described in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, as it can rapidly and specifically detect Mycobacterium abscessus complex 23S rRNA. Among these, a primer set in which the combination of the first and second primers is oligonucleotides consisting of any of the base sequences described in SEQ ID NOs: 14 and 65, 14 and 71, 16 and 65, 16 and 67, 16 and 71, 22 and 65, 22 and 67, 22 and 71, 20 and 65, 20 and 67, 20 and 69, 20 and 73, 18 and 63, 18 and 65, 18 and 69, 12 and 25, 12 and 27, 12 and 29, 12 and 31, 12 and 33, 12 and 35, 12 and 37, 12 and 39, 12 and 41, 12 and 43, 12 and 45, 12 and 47, 12 and 49, 12 and 51, 12 and 53, 12 and 55, 12 and 57, 12 and 59, or 12 and 61 is even more preferred.

[0023] The primer set of the present invention is useful as a primer set for amplifying nucleic acids containing a specific nucleotide sequence of Mycobacterium abscessus complex 23S rRNA or a complementary sequence of said specific nucleotide sequence, using nucleic acid amplification methods commonly used by those skilled in the art, such as RT-PCR. Furthermore, if an RNA polymerase promoter is added to the 5' end of either the first or second primer, nucleic acids containing the specific nucleotide sequence with the RNA polymerase promoter added or a complementary sequence of said specific nucleotide sequence can be synthesized using the RNA polymerase corresponding to the promoter. This is preferable because RNA can then be amplified using methods that amplify RNA at a constant temperature, such as NASBA (Nucleic Acid Sequence Based Amplification), TMA (Transcription-Mediated Amplification), and TRC (Transcription-Reverse transcription Concerted reaction). The promoter attached to the 5' end of the primer should be one that corresponds to the RNA polymerase used for RNA amplification (for example, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, which are commonly used in the field of molecular biology). Furthermore, a transcription start region known to affect transcription efficiency may be added to the promoter. A specific example of a promoter attached to the 5' end of the primer (T7 promoter) when using T7 RNA polymerase for RNA amplification is the oligonucleotide consisting of the sequence described in Sequence ID No. 73.

[0024] To detect Mycobacterium abscessus complex 23S rRNA using the oligonucleotide of the present invention, the procedure may be carried out, for example, by the steps shown in (1) to (6) below. (1) A first primer, which is an oligonucleotide that can specifically hybridize with the base sequence described in Sequence ID No. 10 or its complementary sequence, hybridizes with Mycobacterium abscessus complex 23S rRNA, and an enzyme having RNA-dependent DNA polymerase activity synthesizes cDNA complementary to the specific base sequence, thereby generating an RNA-DNA double helix with the RNA. (2) A step of degrading the RNA of the RNA-DNA double strand with an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA), (3) A step of hybridizing the single-stranded DNA with a second primer which is an oligonucleotide that can specifically hybridize with the base sequence described in Sequence ID No. 23 or its complementary sequence (where an RNA polymerase promoter is attached to the 5' end of either the first or second primer), and generating a double-stranded DNA containing a promoter capable of transcribing RNA of a specific base sequence or a sequence complementary to a specific base sequence using an enzyme having DNA-dependent DNA polymerase activity. (4) A step of producing an RNA transcript using the double-stranded DNA as a template with an enzyme having RNA polymerase activity. (5) A step in which the RNA transcript serves as a template for cDNA synthesis in the reaction of (1) above, thereby generating RNA transcripts in a chain reaction. (6) A step of measuring the amount of RNA transcript over time using an oligonucleotide that contains sequence number 3 or its complementary sequence, wherein the oligonucleotide is hybridizable to a specific base sequence or a sequence complementary to the specific base sequence and is labeled with an intercalator fluorescent dye.

[0025] The enzyme having RNA-dependent DNA polymerase activity used in step (1), the enzyme having RNase H activity used in step (2), and the enzyme having DNA-dependent DNA polymerase activity used in step (3) can each be added separately or in various combinations, but a retrovirus-derived reverse transcriptase possessing the aforementioned activities can also be used. The reverse transcriptase is not particularly limited, but commonly used reverse transcriptases in the field of molecular biology, such as 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, can be used.

[0026] The reaction temperature in the Mycobacterium abscessus complex 23S rRNA detection method described above depends on the heat resistance and activity of each enzyme used, as well as the Tm of the primer / probe. However, if the enzymes used are AMV reverse transcriptase and T7 RNA polymerase, and the primer / probe length is in the range of 14 to 26 bases, the reaction temperature can be set in the range of 35 to 65°C, and more preferably in the range of 40 to 50°C.

[0027] The method for detecting Mycobacterium abscessus complex 23S rRNA according to the above-described embodiment allows for the termination of the measurement at any time when a significant increase in fluorescence is observed, as fluorescence intensity is measured over time. Nucleic acid amplification and measurement can typically be completed within 20 minutes.

[0028] The method for detecting Mycobacterium abscessus complex 23S rRNA according to the above-described embodiment allows for the automatic amplification and detection of Mycobacterium abscessus complex 23S rRNA by adding a sample to a Mycobacterium abscessus complex 23S rRNA reagent containing the first primer, the second primer, and an oligonucleotide labeled with an intercalator fluorescent dye, and placing it on a temperature-controlled block capable of fluorescence detection over time. The oligonucleotide for detecting Mycobacterium abscessus complex 23S rRNA according to the present invention is preferably one consisting of the nucleotide sequences described in SEQ ID NOs: 3 to 8. [Effects of the Invention]

[0029] The oligonucleotide of the present invention hybridizes with a specific sequence (specific base sequence) and a portion of its complementary sequence that are specific to Mycobacterium abscesses complex 23S rRNA, thereby enabling the specific base sequence or complementary sequence of Mycobacterium abscesses complex 23S rRNA to be specifically detected.

[0030] The oligonucleotide of the present invention and the method for detecting Mycobacterium abscesses complex bacteria using the oligonucleotide can rapidly and sensitively detect Mycobacterium abscesses complex bacteria contained in a sample, and have extremely low false positive rates and high specificity. Therefore, it is possible to provide test results to physicians as quickly as possible, which is expected to contribute to preventing the spread of infection and preventing the development of drug-resistant bacteria through the administration of appropriate drugs. [Examples]

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

[0032] Example 1: Standard RNA preparation or dilution of bacterial culture medium Standard RNA of Mycobacteroides abscessus subsp. abscessus, a subspecies of Mycobacterium abscessus complex, and Mycobacteroides abscessus subsp. massiliense, which will be used in the examples described below, were prepared by the method shown below. The prepared standard RNA was quantified based on the absorbance at 260 nm. (1) Mycobacterium abscesses subspecies abscesses standard RNA, a type of Mycobacterium abscesses complex bacterium. Based on the 23S rRNA sequence, a 23S rRNA gene was artificially generated, and after in vitro transcription, the transcript was purified to prepare standard RNA for Mycobacterium abscessus subspecies *Masiliense*. (2) Mycobacterium abscesses subspecies maciliense standard RNA, a type of Mycobacterium abscesses complex bacterium. Based on the 23S rRNA sequence, a 23S rRNA gene was artificially generated, and after in vitro transcription, the transcript was purified to prepare standard RNA for Mycobacterium abscessus subspecies *Masiliense*.

[0033] Example 2 Preparation of oligonucleotides labeled with intercalator fluorescent dyes An oligonucleotide labeled with an intercalator fluorescent dye, as shown in (A) below (hereinafter referred to as an INAF probe), was prepared based on the method disclosed in Japanese Patent Application Publication No. 2000-316587. (A) Oligonucleotides comprising the nucleotide sequence or complementary sequence described in SEQ ID NO: 3, the nucleotide sequence or complementary sequence described in SEQ ID NO: 5, or the nucleotide sequence or complementary sequence described in SEQ ID NO: 7, wherein in SEQ ID NO: 3, thiazole orange is labeled via a linker between the 5th thymine and the 6th adenine from the 5' end, and in SEQ ID NOs: 5 and 7, thiazole orange is labeled via a linker between the 3rd thymine and the 4th adenine from the 5' end.

[0034] Example 3: Investigation of oligonucleotides for detecting Mycobacterium abscesses complex 23S rRNA. The combinations of the first primer, the second primer, and the INAF probe shown in Table 1 (hereinafter referred to as oligonucleotide combinations) were evaluated using the method described below. The INAF probes listed in Table 1 are the probes prepared in Example 2. (1) Of the standard RNAs prepared in Example 1, Mycobacterium abscesses subspecies abscesses standard RNA and Mycobacterium abscesses subspecies masiliense standard RNA (Example 1(1), Example 1(2)) were diluted to 1000 copies / 15 μL using 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) The reaction solution, consisting of the following composition, was dispensed into drying tubes and evaporated.

[0035] Reaction mixture composition: Concentrations are for RNA sample, starting solution, and final concentration after addition (in 30 μL). 60mM Tris-HCl buffer (pH8.35) 300mM Trehalose 0.39mM each dATP, dCTP, dGTP, dTTP 2.1 mM ATP, CTP, UTP 1.5mM GTP 3.2mM ITP 0.2 μM First primer (an oligonucleotide consisting of the base sequence of each SEQ ID NO: 53, with the T7 promoter (SEQ ID NO: 53) added to the 5' end) 0.2 μM Second primer 75nM INAF probe (prepared in Example 2) 0.038 mg / mL Bovine serum albumin 142U T7 RNA polymerase 6.4U AMV reverse transcriptase (3) After the above evaporation drying, 15 μL of RNA sample was added, and the mixture was incubated at 46°C for 5 minutes. Then, 15 μL of an initiation solution consisting of the following composition was added and the mixture was stirred.

[0036] Enzyme solution composition: Final concentration during reaction (in 30 μL) 11.50% Dimethyl Sulfoxide 20.5 mM magnesium chloride 104 mM potassium chloride (4) Subsequently, using a temperature-controlled fluorescence spectrophotometer capable of directly measuring the evaporation tubes, the reaction was carried out at 46°C, and the fluorescence intensity of the reaction solution was measured over time for 20 minutes.

[0037] The time at which the starting solution was added and stirring was completed was defined as 0 minutes. A positive result was determined when the fluorescence intensity ratio of the reaction solution (the fluorescence intensity value at a predetermined time divided by the background fluorescence intensity ratio) exceeded 1.60, and this time was defined as the detection time. The results are shown in Tables 1, 2, and 3. Each combination was measured twice, and the average value was used. "ND" means that the fluorescence intensity ratio 20 minutes after the start of the reaction was 1.60 or less (negative result).

[0038] Regarding the detection performance of Mycobacterium abscesses subspecies abscesses 23S rRNA (Table 1), all of the oligonucleotide combinations A01 to A19, A38, and A39 examined in this example detected Mycobacterium abscesses subspecies abscesses 23S rRNA at 1000 copies / test within 20 minutes. In particular, oligonucleotide combinations A38 and A39 detected Mycobacterium abscesses subspecies abscesses 23S rRNA at 1000 copies / test within an average of 5 minutes, indicating that these oligonucleotide combinations can rapidly detect Mycobacterium abscesses subspecies abscesses 23S rRNA.

[0039] Regarding the detection performance of Mycobacterium abscesses subspecies masiliense 23S rRNA (Table 1), all of the oligonucleotide combinations A20 to A39 examined in this example detected Mycobacterium abscesses subspecies masiliense 23S rRNA at a rate of 1000 copies / test within an average of 5 minutes. Therefore, these oligonucleotide combinations can be considered capable of rapidly detecting Mycobacterium abscesses subspecies masiliense 23S rRNA.

[0040] [Table 1]

[0041] The Mycobacterium abscessus complex has three subspecies: Mycobacterium abscessus subspecies abscessus, Mycobacterium abscessus subspecies boretti, and Mycobacterium abscessus subspecies masiliensis.

[0042] Among the oligonucleotide combinations examined in this example, A39 was selected from those with a detection time of 5 minutes or less to detect all subspecies of Mycobacterium abscessus complex (Table 2) and to perform cross-reactivity (Table 3).

[0043] For the detection of Mycobacterium abscesses complex bacteria, we detected Mycobacterium abscesses subspecies abscesses, Mycobacterium abscesses subspecies boretti, or Mycobacterium abscesses subspecies maciliensis at a concentration of 2 CFU / test. Cross-reactivity was assessed at 2 × 10⁻⁶. 5 The CFU / test failed to detect 20 species of non-tuberculous mycobacteria and demonstrated high specificity for Mycobacterium abscessus complex. Therefore, the oligonucleotide combination investigated in this example can be considered a combination of oligonucleotides capable of specifically detecting the 23S rRNA of Mycobacterium abscessus complex.

[0044] [Table 2]

[0045] [Table 3]

Claims

1. An oligonucleotide for detecting Mycobacterium abscesses complex bacteria, characterized in that it comprises a specific nucleotide sequence or complementary sequence of 23S rRNA or 23S rDNA of Mycobacterium abscesses complex bacteria, and consists of the nucleotide sequence described in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7.

2. The oligonucleotide according to claim 1, characterized in that the oligonucleotide is labeled with a fluorescent dye and configured to change its fluorescence properties when it forms complementary double helixes.

3. The oligonucleotide according to claim 2, characterized in that the oligonucleotide is labeled with an intercalator fluorescent dye.

4. A method for detecting Mycobacterium abscesses complex bacteria, characterized by using an oligonucleotide described in any one of claims 1 to 3, for detecting a specific base sequence or complementary sequence of 23S rRNA or 23S rDNA of Mycobacterium abscesses complex bacteria.

5. A method for detecting Mycobacterium abscesses complex bacteria according to claim 4, characterized by comprising the step of amplifying a specific base sequence or its complementary sequence using a set of primers.

6. The method for detecting Mycobacterium abscesses complex bacteria according to claim 4 or 5, characterized in that the set of primers comprises a first primer that hybridizes to the sequence described in Sequence ID No. 9 and a second primer that hybridizes to the sequence described in Sequence ID No.

24.

7. A method for detecting Mycobacterium abscesses complex bacteria according to any one of claims 4 to 6, characterized in that the first primer consists of 16 to 26 consecutive bases in the sequence or complementary sequence described in Sequence ID No. 9, and the second primer consists of 19 to 26 consecutive bases in the sequence or complementary sequence described in Sequence ID No.

24.

8. A reagent kit for detecting Mycobacterium abscesses complex, comprising an oligonucleotide according to any one of claims 1 to 3 and a primer set according to claim 6 or 7.

9. The kit according to claim 8, comprising a first primer having the sequence described in Sequence ID No. 12 and a second primer having the sequence described in Sequence ID No. 25.

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