Oligonucleotide for identifying genus mycobacterium and application of the same

Abstract

To provide a useful method for detecting the genus Mycobacterium acid-fast bacterium species.SOLUTION: A method for detecting the genus Mycobacterium acid-fast bacterium species that can be included in a sample includes amplifying a plurality of nucleic acid regions derived from a multicopy gene of the genus Mycobacterium acid-fast bacterium species, and the multicopy gene has the following characteristics (a) and / or (b): (a) the sequence identity of the genus Mycobacterium acid-fast bacterium species of a detection target between each strain is 82% or more, and the sequence identity between the genus Mycobacterium acid-fast bacterium species of a detection target and the other genus Mycobacterium acid-fast bacterium species is less than 82%; and (b) a multicopy gene is present in more than 98% of strains of the genus Mycobacterium acid-fast bacterium species of a detection target.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to oligonucleotides and the like for detecting (particularly differentiating) acid-fast bacterium of the genus Mycobacterium, represented by Mycobacterium tuberculosis (MTB), Mycobacterium avium (MAV), Mycobacterium intracellulare (MIN), Mycobacterium abscessus (MAB), and Mycobacterium kansasii (MKA), which may be contained in a sample. Furthermore, the present invention relates to a method for detecting (particularly differentiating) acid-fast bacteria of the genus Mycobacterium, such as Mycobacterium tuberculosis (MTB), Mycobacterium avium (MAV), Mycobacterium intracellulare (MIN), Mycobacterium abscessus (MAB), and Mycobacterium kansasii (MKA), which may be contained in a sample, using the oligonucleotide, as well as reagents and kits for use in the method. [Background technology]

[0002] Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), and is highly pathogenic and can cause human-to-human infection. Nontuberculous mycobacteria (NTM), including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium kansasii, are known to cause opportunistic infections in immunocompromised patients, such as those with NTM disease or AIDS. Because the clinical symptoms of TB and NTM disease are similar, rapid and accurate differentiation of the causative pathogen, Mycobacterium spp., is of great public health importance.

[0003] Culture identification methods have long been used as a method for testing Mycobacterium acid-fast bacteria. However, they generally grow slowly, and it often takes about a month or more to identify the bacterial species. For this reason, rapid testing using nucleic acid amplification methods has been conducted in recent years. The main nucleic acid amplification methods used are PCR, real-time PCR, and TRC (patent document 1, patent document 2, patent document 3, patent document 4). Examples of nucleic acids targeted by nucleic acid amplification methods include ribosomal RNA genes and rpoB genes.

[0004] It is known that the gene sequences of Mycobacterium species are very similar among them. For this reason, the gene sequences that have been targeted by conventional nucleic acid amplification methods often result in false positives due to cross-reactions, which is an issue (Non-Patent Documents 1 and 2). Furthermore, only one copy of these gene sequences exists per mycobacterial genome.

[0005] Nucleic acid amplification methods that target gene sequences known as multicopy sequences, in which multiple copies of a specific base sequence with high sequence identity exist on the genome, are also known. For example, the IS6110 gene of Mycobacterium tuberculosis is a multicopy gene and is widely used as a target for nucleic acid amplification methods (Non-Patent Document 3). Meanwhile, non-tuberculous mycobacteria also contain multicopy genes, such as the IS1245 gene of Mycobacterium avium. However, some subspecies may not be detected, and species-specific detection may not be possible (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2021 / 124960 [Patent Document 2] Patent No. 6221563 [Patent Document 3] Patent No. 6387593 [Patent Document 4] Patent No. 5286996 [Non-patent literature]

[0007] [Non-Patent Document 1] Kinuyo Chikamatsu et al., Tuberculosis, 2016, 91, 623-629 [Non-patent document 2] Fujimori Takumi et al., Medical Testing, 2020, 69, 145-151 [Non-patent document 3] Thierry, D. et.al., J. Clinical. MicroBiol.,1990,28,2668-2673 [Non-patent document 4] Ichikawa, K. et.al., J. Med. Microbiol., 2009, 58, 945-950 [Non-Patent Document 5] Park,J.et.al.,Microbiol Spectr.,2023,11,e0160623 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention was made with the aim of developing a useful method for detecting (particularly differentiating) Mycobacterium mycobacteria by targeting a novel gene sequence that is completely different from the gene sequences that have been targeted in the past.

[0009] One object of the present invention is to provide a useful method for detecting (particularly differentiating) Mycobacterium mycobacteria, in particular a method for simultaneously detecting Mycobacterium mycobacteria with specificity, high sensitivity, and completeness while targeting multiple novel gene sequences present in the genome of Mycobacterium mycobacteria. [Means for solving the problem]

[0010] As a result of intensive research in light of the above-mentioned problems, the present inventors have found that by using a probe in which only one end of an oligonucleotide having a specific base sequence is labeled, it is possible to detect (particularly simultaneously detect in one reaction) and particularly to differentiate Mycobacterium acid-fast bacteria. Based on this finding, the present inventors have conducted further research and have completed the present invention.

[0011] The present invention typically includes the aspects described in the following paragraphs. [Section 1] 1. A method for detecting Mycobacterium spp. that may be contained in a sample, the method comprising amplifying multiple nucleic acid regions derived from a multicopy gene of Mycobacterium spp., wherein the multicopy gene has the following characteristics (a) and / or (b): (a) the sequence identity between strains of the Mycobacterium species to be detected is 82% or more, and the sequence identity between the Mycobacterium species to be detected and other Mycobacterium species is less than 82%; (b) present in more than 98% of strains of Mycobacterium species detected. [Section 2] Item 1, wherein the multicopy gene has characteristics (a) and (b). [Section 3] Item 3. The method according to Item 1 or 2, wherein the plurality of nucleic acid regions each independently have a length of 50 to 1500 bases. [Section 4] Item 4. The method according to any one of Items 1 to 3, wherein the sequence identity of the plurality of nucleic acid regions is 85% or more. [Section 5] Item 5. The method according to any one of Items 1 to 4, wherein the multicopy gene is an HNH nuclease gene. [Section 6] 6. The method according to any one of Items 1 to 5, wherein each of the plurality of nucleic acid regions shows 85% or more sequence identity to any of the nucleotide sequence shown in SEQ ID NO: 1 or a complementary nucleotide sequence thereof, the nucleotide sequence shown in SEQ ID NO: 3 or a complementary nucleotide sequence thereof, the nucleotide sequence shown in SEQ ID NO: 5 or a complementary nucleotide sequence thereof, the nucleotide sequence shown in SEQ ID NO: 7 or a complementary nucleotide sequence thereof, and the nucleotide sequence shown in SEQ ID NO: 9 or a complementary nucleotide sequence thereof. [Section 7] 7. The method according to any one of Items 1 to 6, wherein each of the plurality of nucleic acid regions shows 85% or more sequence identity to any of the nucleotide sequence shown in SEQ ID NO: 2 or a complementary nucleotide sequence thereof, the nucleotide sequence shown in SEQ ID NO: 4 or a complementary nucleotide sequence thereof, the nucleotide sequence shown in SEQ ID NO: 6 or a complementary nucleotide sequence thereof, the nucleotide sequence shown in SEQ ID NO: 8 or a complementary nucleotide sequence thereof, and the nucleotide sequence shown in SEQ ID NO: 10 or a complementary nucleotide sequence thereof. [Section 8] 8. The method according to any one of Items 1 to 7, wherein the Mycobacterium species is one or more of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium kansasii. [Section 9] A probe for detecting Mycobacterium genus acid-fast bacteria, having any one of the following characteristics (A) to (E) and the following characteristic (F): (A) a base sequence A1 of at least 10 consecutive bases in the base sequence of 50 to 90 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence A2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence A1; (B) a base sequence B1 of at least 10 consecutive bases in the base sequence of positions 30 to 70 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence B2 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence B1; (C) a base sequence C1 of at least 10 consecutive bases in the base sequence of positions 45 to 85 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence C2 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence C1; (D) a base sequence D1 of at least 10 consecutive bases in the base sequence of positions 45 to 85 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence D2 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence D1; (E) a base sequence E1 of at least 10 consecutive bases in the base sequence of positions 45 to 85 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence E2 in which 1 to 3 bases have been substituted, deleted, inserted, or added in the base sequence E1; (F) Only either the 5' or 3' end is labeled. [Section 10] Item 10. The probe according to Item 9, wherein the length of the base sequences (A) to (E) is 12 to 20 bases. [Section 11] Item 11. The probe according to Item 9 or 10, wherein the base sequence of (A) comprises the base sequence shown in SEQ ID NO: 11 or a complementary base sequence thereof, the base sequence of (B) comprises the base sequence shown in any one of SEQ ID NOs: 14, 33, 34, 35, and 36 or a complementary base sequence thereof, the base sequence of (C) comprises the base sequence shown in any one of SEQ ID NOs: 17, 37, 38, 39, 40, 41, and 42 or a complementary base sequence thereof, the base sequence of (D) comprises the base sequence shown in SEQ ID NO: 20 or a complementary base sequence thereof, and the base sequence of (E) comprises the base sequence shown in SEQ ID NO: 24 or a complementary base sequence thereof. [Section 12] The probe according to any one of Items 9 to 11, wherein the label (F) is a fluorescent dye label, preferably a label with a fluorescent quenching dye that is quenched (particularly, quenched by interaction with guanine) when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe, and more preferably, the labeled terminal base is cytosine. [Section 13] Item 13. The probe according to any one of Items 9 to 12, wherein the label (F) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives, and is preferably a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Section 14] A method for detecting Mycobacterium species that may be contained in a sample, using one or more probes according to any one of Items 9 to 13. [Section 15] The following steps (1), (2), and (3): (1) providing a sample that may contain Mycobacterium species; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using the one or more probes; Item 15. The method according to Item 14, comprising: [Section 16] Item 16. The method according to Item 15, wherein the Mycobacterium species is one or more of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium kansasii. [Section 17] Item 17. The method according to Item 15 or 16, wherein the step (2) is carried out by PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B. [Section 18] Item 18. The method according to Item 17, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Section 19] 19. The method according to any one of Items 15 to 18, wherein the step (2) is carried out using a primer set having any one of the following characteristics (G) to (K): (G) a first primer having a base sequence S1 of at least 20 consecutive bases in the base sequence of positions 1 to 65 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1; and a second primer having a base sequence S3 of at least 20 consecutive bases in the base sequence of positions 65 to 130 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second primer is complementary to a DNA extension product of the first primer; (H) a first primer having a base sequence S5 of at least 20 consecutive bases in the base sequence of positions 1 to 60 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S5; and a second primer having a base sequence S7 of at least 20 consecutive bases in the base sequence of positions 60 to 120 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second primer is complementary to a DNA extension product of the first primer; (I) a first primer having a base sequence S9 of at least 20 consecutive bases in the base sequence of positions 1 to 60 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence S10 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S9; and a second primer having a base sequence S11 of at least 20 consecutive bases in the base sequence of positions 60 to 120 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence S12 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S11, wherein the second primer is complementary to a DNA extension product of the first primer; (J) a first primer having a base sequence S13 of at least 20 consecutive bases in the base sequence of positions 1 to 60 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S14 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S13; and a second primer having a base sequence S15 of at least 20 consecutive bases in the base sequence of positions 60 to 114 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S16 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S15, wherein the second primer is complementary to a DNA extension product of the first primer; (K) A first primer having a base sequence S17 of at least 20 consecutive bases in the base sequence from positions 1 to 65 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence S18 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S17; and a second primer having a base sequence S19 of at least 20 consecutive bases in the base sequence from positions 65 to 130 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence S20 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S19, wherein the second primer is complementary to a DNA extension product of the first primer. [Section 20] the primer set having the characteristic (G) comprises the first primer having the nucleotide sequence shown in SEQ ID NO: 12, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added, and the second primer having the nucleotide sequence shown in SEQ ID NO: 13, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added; the primer set having the characteristic (H) comprises the first primer having a nucleotide sequence shown in SEQ ID NO: 15 or 43, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 nucleotides have been substituted, deleted, inserted or added, and the second primer having a nucleotide sequence shown in any of SEQ ID NOs: 16, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 nucleotides have been substituted, deleted, inserted or added; the primer set having the characteristic (I) comprises the first primer having a nucleotide sequence shown in any one of SEQ ID NOs: 18, 55, 56, 57, 58, 59, 60, 61, 62, and 63, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substitution, deletion, insertion, or addition of 1 to 3 nucleotides in said nucleotide sequence, and the second primer having a nucleotide sequence shown in SEQ ID NO: 19 or 64, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substitution, deletion, insertion, or addition of 1 to 3 nucleotides in said nucleotide sequence; the primer set having the characteristic (J) comprises the first primer having the nucleotide sequence shown in SEQ ID NO: 21, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added, and the second primer having the nucleotide sequence shown in SEQ ID NO: 22 or 23, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added; Item 20. The method according to Item 19, wherein the primer set having characteristic (K) comprises the first primer having the nucleotide sequence shown in SEQ ID NO: 25, a complementary nucleotide sequence thereof, or a nucleotide sequence of either of these nucleotide sequences in which 1 to 3 bases have been substituted, deleted, inserted, or added; and the second primer having the nucleotide sequence shown in SEQ ID NO: 26, a complementary nucleotide sequence thereof, or a nucleotide sequence of either of these nucleotide sequences in which 1 to 3 bases have been substituted, deleted, inserted, or added. [Section 21] 21. The method according to any one of Items 15 to 20, wherein the detecting step in step (3) is carried out by melting curve analysis. [Section 22] A reagent or kit for detecting Mycobacterium species, for use in the method according to any one of Items 1 to 8 and Items 14 to 21. [Section 24] A reagent or kit for detecting Mycobacterium species, comprising at least one of the probes according to any one of Items 9 to 13 and / or at least one of the primer sets according to Item 19 or 20. [Effects of the Invention]

[0012] According to the present invention, Mycobacterium acid-fast bacteria can be detected (particularly differentiated) accurately and with high specificity and high detection sensitivity in a short time, for example, and can make a great contribution to the field of clinical diagnosis. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of Test Example 4, in which 100 copies of Mycobacterium tuberculosis DNA were detected. [Figure 2] FIG. 1 shows the results of Test Example 5, in which 100 copies of Mycobacterium avium DNA were detected. [Figure 3] FIG. 1 shows the results of Test Example 6, in which 100 copies of Mycobacterium intracellulare DNA were detected. [Figure 4]FIG. 1 shows the results of Test Example 7, in which 100 copies of Mycobacterium abscessus DNA were detected. [Figure 5] FIG. 1 shows the results of Test Example 8, in which 100 copies of Mycobacterium kansasii DNA were detected. [Figure 6] 1 shows the design of primers and probes for Mycobacterium tuberculosis. [Figure 7] Design of primers and probes for Mycobacterium avium. [Figure 8] Schematic diagram of primer and probe designs for Mycobacterium intracellulare. [Figure 9] Design of primers and probes for Mycobacterium abscessus. [Figure 10] 1 shows the design of primers and probes for Mycobacterium kansasii. [Figure 11] FIG. 1 shows the results of Test Example 10, in which 20 copies of Mycobacterium avium DNA were detected. [Figure 12] FIG. 1 shows the results of Test Example 10, in which 20 copies of Mycobacterium intracellulare DNA were detected. [Figure 13] Design of primers and probes for Mycobacterium avium. [Figure 14] Schematic diagram of primer and probe designs for Mycobacterium intracellulare. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in more detail below by showing embodiments of the present invention, but the present invention is not limited to these. All non-patent and patent documents described in this specification are incorporated herein by reference in their entirety. In addition, the term "~" in this specification means "at least, at most," and for example, if the specification states "X~Y," it means "at least X and at most Y." In this specification, "and / or" means any one or any possible combination of two or more of the listed elements. In this specification, "comprising" encompasses the concepts of "consisting essentially of" and "consisting only of."

[0015] In this specification, a nucleic acid primer may be simply referred to as a primer, and a nucleic acid probe and a labeled probe may be simply referred to as a probe, and these are also collectively referred to as oligonucleotides.

[0016] As used herein, "multicopy" refers to the presence of two or more copies of a specific highly identical base sequence in the genomic DNA of a certain species, whereas "single copy" refers to the presence of only one copy of a specific base sequence in the genomic DNA of a certain species.

[0017] The multiple nucleic acid regions derived from the multicopy gene each independently have a length of, for example, 10 to 1500 bases, preferably 50 to 1500 bases, more preferably 50 to 1000 bases, even more preferably 50 to 500 bases, and even more preferably 50 to 200 bases.

[0018] The number of nucleic acid regions derived from multicopy genes present in genomic DNA is not particularly limited as long as it is two or more, and may be, for example, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 3 to 5. Furthermore, some differences in the base sequences of the multiple nucleic acid regions derived from multicopy genes are acceptable. The sequence identity (hereinafter sometimes simply referred to as "identity") of the multiple nucleic acid regions is typically 70% or more, preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more. Targeting the multiple nucleic acid regions enables detection of Mycobacterium mycobacteria with higher sensitivity than targeting a nucleic acid region derived from a single-copy gene.

[0019] In one embodiment, the present invention is characterized by detecting a multicopy gene of Mycobacterium species, which may have, but is not limited to, the following characteristics (a) and / or (b): (a) The sequence identity between strains of the Mycobacterium species to be detected is 82% or more, and the sequence identity between the Mycobacterium species to be detected and other Mycobacterium species is less than 82%. (b) present in more than 98% of strains of Mycobacterium species detected.

[0020] In feature (a), the sequence identity between strains of the Mycobacterium species to be detected is preferably 83% or more, more preferably 84% or more, and even more preferably 85% or more. Furthermore, the sequence identity between the Mycobacterium species to be detected and other Mycobacterium species need only be lower than the sequence identity between strains of the Mycobacterium species to be detected, and may be less than 83%, less than 84%, or less than 85%.

[0021] When the Mycobacterium species to be detected is Mycobacterium tuberculosis, the multicopy gene may be the RVBD_0094c gene of the H37Rv strain (SEQ ID NO: 1) or a partial sequence thereof (e.g., SEQ ID NO: 2), and the sequence identity between the H37Rv strain and other strains is 82% or more, preferably 83% or more, more preferably 84% or more, even more preferably 85% or more, and may be 86% or more, 87% or more, 88% or more, or 89% or more. In addition, the sequence identity between Mycobacterium tuberculosis (H37Rv strain) and other Mycobacterium species (e.g., any strain of Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellulare, or Mycobacterium abscessus) may be less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, or less than 82%, or may be 81% or less, 80% or less, 79% or less, or 78% or less.

[0022] When the Mycobacterium species to be detected is Mycobacterium avium, the multicopy gene may be the MAA44156_02837 gene (sequence number 3) of subsp. avium strain DSM 44156 (hereinafter simply referred to as "DSM 44156 strain") or a partial sequence thereof (e.g., sequence number 4), and the sequence identity between DSM 44156 strain and other strains is 82% or more, preferably 83% or more, more preferably 84% or more, even more preferably 85% or more, and may be 86% or more, 87% or more, 88% or more, or 89% or more. In addition, the sequence identity between Mycobacterium avium (strain DSM 44156) and other Mycobacterium species (e.g., any strain of Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium tuberculosis, or Mycobacterium abscessus) may be less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, or less than 82%, or may be 81% or less, 80% or less, or 79% or less.

[0023] When the Mycobacterium species to be detected is Mycobacterium intracellulare, the multicopy gene may be the KN251_15665 gene (SEQ ID NO: 5) of the ATCC 13950 strain or a partial sequence thereof (e.g., SEQ ID NO: 6), and the sequence identity between the ATCC 13950 strain and other strains may be 82% or more, preferably 83% or more, more preferably 84% or more, even more preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, or 92% or more. Additionally, the sequence identity between Mycobacterium intracellulare (ATCC 13950 strain) and other Mycobacterium species (e.g., any strain of Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium kansasii, or Mycobacterium abscessus) may be less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, or less than 82%, or may be 81% or less, 80% or less, or 79% or less.

[0024] When the Mycobacterium species to be detected is Mycobacterium abscessus, the multicopy gene may be the MAB_0990c gene (SEQ ID NO: 7) of the ATCC 19977 strain or a partial sequence thereof (e.g., SEQ ID NO: 8), and the sequence identity between the ATCC 19977 strain and other strains is 82% or more, preferably 83% or more, more preferably 84% or more, and even more preferably 85% or more. In addition, the sequence identity between Mycobacterium abscessus (ATCC 19977 strain) and other Mycobacterium species (e.g., any strain of Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium avium, or Mycobacterium tuberculosis) may be less than 85%, less than 84%, less than 83%, or less than 82%, or may be 81% or less, 80% or less, or 79% or less.

[0025] When the Mycobacterium species to be detected is Mycobacterium kansasii, the multicopy gene may be the MKAN_05295 gene (SEQ ID NO: 9) of the ATCC 12478 strain or a partial sequence thereof (e.g., SEQ ID NO: 10), and the sequence identity between the ATCC 12478 strain and other strains is 82% or more, preferably 83% or more, more preferably 84% or more, even more preferably 85% or more, and may be 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Additionally, the sequence identity between Mycobacterium kansasii (ATCC 12478 strain) and other Mycobacterium species (e.g., any strain of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, or Mycobacterium abscessus) may be less than 85%, less than 84%, less than 83%, or less than 82%, or may be 81% or less.

[0026] It is preferable that the multicopy gene of the Mycobacterium species to be detected in feature (b) is present in more than 98% of the strains, more preferably 98.5% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more.

[0027] Methods for confirming the presence of multicopy genes in Mycobacterium mycobacterium strains include, but are not limited to, database searches and measurements of clinical specimens, culture fluids, and clinical isolates. Database searches involve searching for the base sequence of the multicopy gene in a database such as Blast (Basic Local Alignment Search Tool), and the percentage of hits among the complete genomes registered for the target Mycobacterium mycobacterium species is calculated. Measurement of clinical specimens, culture fluids, and clinical isolates involves calculating the percentage of samples derived from the target Mycobacterium mycobacterium identified by other testing methods in which the multicopy gene was detected by genetic testing.

[0028] In one embodiment, the multicopy gene preferably has at least characteristic (b), and more preferably has characteristics (a) and (b).

[0029] Each of the multiple nucleic acid regions derived from a multicopy gene preferably exhibits, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity to any of the nucleotide sequence shown in SEQ ID NO: 1 or its complementary nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 3 or its complementary nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 5 or its complementary nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 7 or its complementary nucleotide sequence, and the nucleotide sequence shown in SEQ ID NO: 9 or its complementary nucleotide sequence. Furthermore, each of the multiple nucleic acid regions derived from a multicopy gene preferably exhibits, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity to any of the nucleotide sequence shown in SEQ ID NO: 2 or its complementary nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 4 or its complementary nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 6 or its complementary nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 8 or its complementary nucleotide sequence, and the nucleotide sequence shown in SEQ ID NO: 10 or its complementary nucleotide sequence.

[0030] The type of multicopy gene is not particularly limited, and may be a housekeeping gene, a functional gene, or a gene without a function. The multicopy gene is preferably not an insertion sequence (IS) gene, but is preferably a nuclease gene, more preferably an endonuclease gene, and even more preferably an HNH endonuclease gene. An IS gene is a short DNA that acts as a simple transcription factor, and refers to a gene whose name includes an IS and a number.

[0031] The HNH endonuclease gene as used herein is characterized by encoding a nuclease consisting of 30 to 40 amino acids and having an HNH motif consisting of His-Asn-His, and by containing a multicopy gene. The multicopy gene contained in this gene is different from an insertion sequence (IS) and exists as a structural gene encoding a protein, with 2 to 10 copies contained in the genome of Mycobacterium mycobacteria. Specifically, as shown in SEQ ID NOS: 1, 3, 5, 7, and 9, the sequence of the multicopy gene is conserved among various species, and detection of this sequence enables species-specific and highly sensitive analysis.

[0032] In one embodiment, the present invention provides a method for detecting Mycobacterium species with specificity and high sensitivity in a short time by using a labeled probe having a specific base sequence. The specific base sequence preferably targets a sequence that is specifically present in Mycobacterium species. The detection target may be any Mycobacterium species, and is not particularly limited. For example, M. tuberculosis, also referred to as the M. tuberculosis complex, includes the variants M. tuberculosis var. tuberculosis, M. tuberculosis var. africanum, M. tuberculosis var. bovis (including M. tuberculosis var. bovis BCG strain), M. tuberculosis var. canetti, M. tuberculosis var. caprae, and M. tuberculosis var. microti. tuberculosis var. microti, Mycobacterium tuberculosis variant pinnipedii (M. tuberculosis var. pinnipedii), etc.Mycobacterium avium (M. avium) includes the subspecies M. avium subsp. avium, M. avium subsp. silvaticum, M. avium subsp. paratuberculosis, and M. avium subsp. hominissuis. Mycobacterium intracellulare (M. intracellulare) includes the subspecies M. intracellulare subsp. intracellulare, M. intracellulare subsp. chimaera, and M. intracellulare subsp. yongonense. Mycobacterium abscessus (M. abscessus) includes the subspecies M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Mycobacterium kansasii is classified into subtypes 1–7 and was previously referred to as the M. kansasii complex. However, since different subtypes are now classified as separate species, only subtype 1 is referred to as M. kansasii.The present invention is based on the discovery that Mycobacterium species can be reliably detected, for example, by melting curve analysis, by designing a labeled probe that targets a specific region in the genomic DNA of Mycobacterium species.

[0033] As used herein, SEQ ID NO: 1 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium tuberculosis, specifically the RVBD_0094c gene of Mycobacterium tuberculosis H37Rv strain. SEQ ID NO: 3 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium avium, specifically the MAA44156_02837 gene of Mycobacterium avium subsp. avium strain DSM 44156. SEQ ID NO: 5 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium intracellulare, specifically the KN251_15665 gene of Mycobacterium intracellulare ATCC 13950 strain. SEQ ID NO: 7 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium abscessus, specifically the MAB_0990c gene of Mycobacterium abscessus ATCC 19977 strain. SEQ ID NO: 9 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium kansasii, specifically the MKAN_05295 gene of Mycobacterium kansasii ATCC 12478 strain.

[0034] Furthermore, in this specification, SEQ ID NO: 2 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium tuberculosis, specifically a partial sequence of the RVBD_0094c gene of the Mycobacterium tuberculosis H37Rv strain. SEQ ID NO: 4 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium avium, specifically a partial sequence of the MAA44156_02837 gene of Mycobacterium avium subsp. avium strain DSM 44156. SEQ ID NO: 6 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium intracellulare, specifically a partial sequence of the KN251_15665 gene of the Mycobacterium intracellulare ATCC 13950 strain. SEQ ID NO: 8 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium abscessus, specifically a partial sequence of the MAB_0990c gene of Mycobacterium abscessus ATCC 19977. SEQ ID NO: 10 is a nucleotide sequence corresponding to the genomic DNA sequence of Mycobacterium kansasii, specifically a partial sequence of the MKAN_05295 gene of Mycobacterium kansasii ATCC 12478.

[0035] In the present invention, it is also preferable to use a labeled probe having a specific base sequence that targets a specific region of at least one of the base sequences shown in SEQ ID NOs: 1, 3, 5, 7, and 9, preferably a specific region of at least one of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, and 10. Furthermore, by using a probe set that combines two or more of these labeled probes, it may be possible to detect (particularly differentiate) Mycobacterium species with higher sensitivity and accuracy.

[0036] In one aspect, the present invention provides a probe for detecting Mycobacterium species (specifically, a probe for detecting at least one of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium kansasii) having any of the following characteristics (A) to (E) and characteristic (F): (A) a base sequence A1 of at least 10 consecutive bases (preferably 10 to 25 bases) in the base sequence shown in SEQ ID NO: 1, preferably the base sequence shown in SEQ ID NO: 2, more preferably the base sequence of 50 to 90 bases of SEQ ID NO: 2, or a complementary base sequence thereof, or a base sequence A2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence A1; (B) a base sequence B1 of at least 10 consecutive bases (preferably 10 to 25 bases) in the base sequence shown in SEQ ID NO: 3, preferably the base sequence shown in SEQ ID NO: 4, more preferably the base sequence of positions 30 to 70 of SEQ ID NO: 4, or a complementary base sequence thereof, or a base sequence B2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence B1; (C) a base sequence C1 of at least 10 consecutive bases (preferably 10 to 25 bases) in the base sequence shown in SEQ ID NO: 5, preferably the base sequence shown in SEQ ID NO: 6, more preferably the base sequence of positions 45 to 85 of SEQ ID NO: 6, or a complementary base sequence thereof, or a base sequence C2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence C1; (D) a base sequence D1 of at least 10 consecutive bases (preferably 10 to 25 bases) in the base sequence shown in SEQ ID NO: 7, preferably the base sequence shown in SEQ ID NO: 8, more preferably the base sequence of positions 45 to 85 of SEQ ID NO: 8, or a complementary base sequence thereof, or a base sequence D2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence D1; (E) a base sequence E1 of at least 10 consecutive bases (preferably 10 to 25 bases) in the base sequence shown in SEQ ID NO: 9, preferably the base sequence shown in SEQ ID NO: 10, more preferably the base sequence of positions 45 to 85 of SEQ ID NO: 10, or a complementary base sequence thereof, or a base sequence E2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence E1; (F) Only one of the terminal bases, either the 5'-end or the 3'-end, is labeled (preferably, the terminal base is cytosine, and only the cytosine at the terminal base is labeled with a fluorescent dye). One of such probes for detecting Mycobacterium species may be selected and used, or it is also preferable to use two or more of them in combination.

[0037] One embodiment of the present invention is a method for detecting Mycobacterium species that may be contained in a sample. The method uses a labeled probe having a specific base sequence as described above. In a specific embodiment, by using one specific labeled probe or a combination of the specific labeled probes (probe set) in a reaction solution, Mycobacterium species that may be contained in a sample can be detected with high sensitivity, for example, by PCR-melting curve analysis (including RT-PCR-melting curve analysis).

[0038] In a particular embodiment, the method of the present invention is characterized in that it comprises at least the following steps (i) and (ii): (i) performing a nucleic acid amplification reaction using one or more nucleic acid primer sets having specific base sequences to generate one or more nucleic acid amplification products using a specific region present in Mycobacterium species as a template; and (ii) detecting one or more amplification products obtained in the step (i) using one or more labeled probes or probe sets of the present invention having the specific base sequence. Preferably, the method is a PCR-melting curve analysis method in which a PCR reaction is performed in step (i) and a melting curve analysis is performed in step (ii). Steps (i) and (ii) may be performed in the same reaction solution. Alternatively, the steps may be performed consecutively or simultaneously.

[0039] In a specific embodiment, a method for detecting Mycobacterium species that may be contained in a sample (particularly a specimen sample) preferably comprises at least the following steps (1), (2), and (3): (1) providing a sample that may contain Mycobacterium species; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) A step of detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using the one or more probes or probe sets. In this method, step (2) is preferably carried out by PCR reaction, and step (3) is preferably carried out by melting curve analysis (PCR-melting curve analysis method). Steps (2) and (3) may be carried out in the same reaction solution.

[0040] [Process (1)] In one embodiment, step (1) involves preparing a sample that may contain Mycobacterium species (e.g., biological samples such as body fluids, excrement, and cells; environmental samples such as wipes from facility walls, floors, equipment, fixtures, toilets, etc., or washings used to clean such materials). The sample that can be used in the present invention is not particularly limited as long as it may contain Mycobacterium species. Examples of samples that may contain Mycobacterium species include, but are not limited to, sputum, blood, urine, pus, cerebrospinal fluid, pleural effusion, ascites, gastric fluid, pharyngeal swab, nasal swab, saliva, oral scraping, bronchial lavage fluid, alveolar lavage fluid, tissue slice, skin, vomit, and feces collected from a subject suspected of infection with Mycobacterium species, as well as isolated culture colonies and liquid cultures that may contain Mycobacterium species. Furthermore, each biological sample may be subjected to pretreatment or nucleic acid extraction, such as dilution or suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, alkali treatment, organic solvent treatment, crushing treatment, or grinding treatment, if necessary.

[0041] The method of collecting and preparing the sample is not particularly limited, and known methods can be used depending on the type and purpose of the sample.

[0042] The method for nucleic acid extraction is not particularly limited, and known methods can be used depending on the type of sample and the purpose. For nucleic acid extraction, for example, kits sold by various manufacturers may be used. Alternatively, an automatic extraction and purification device may be used.

[0043] In certain preferred embodiments, the sample may be one that has been prepared without the nucleic acid purification step, which is generally considered essential in conventional nucleic acid amplification reactions. Nucleic acid purification requires specialized reagents and is problematic in that the process is cumbersome, time-consuming, and labor-intensive. Using a sample that has been prepared without the nucleic acid purification step can shorten the time from sample collection to obtaining genetic testing results. For example, the time from sample collection to obtaining genetic testing results can be reduced to within one day, preferably within half a day, more preferably within six hours, even more preferably within three hours, and especially preferably within two hours (e.g., within one hour). Thus, when the method of the present invention is performed using a sample that has not undergone a nucleic acid purification step, the time required for nucleic acid purification can be eliminated, and Mycobacterium species can be detected simply and quickly.

[0044] [Process (2)] In one embodiment, step (2) is preferably a step of generating a nucleic acid amplification product by a nucleic acid amplification method (carrying out a nucleic acid amplification reaction using one or more nucleic acid primer sets). Nucleic acid amplification is a technique for amplifying a few copies of a target nucleic acid to a level at which it can be visualized, i.e., hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in fields such as clinical diagnosis, food hygiene testing, and environmental testing. Examples of such nucleic acid amplification methods include PCR (including RT-PCR), LAMP, LCR, TMA, SDA, RT-LAMP, NASBA, TRC, and TMA. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably PCR (including RT-PCR), but is not limited to this.

[0045] (PCR reaction) PCR is a reaction catalyzed primarily by DNA polymerase. PCR typically involves three steps: (i) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (ii) annealing of a primer to a single-stranded template DNA, and (iii) extension of the primer using a DNA polymerase, with each cycle being repeated. Examples of DNA polymerases include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, and DEEPVENT, as well as their variants. In the present invention, a DNA polymerase belonging to Family B is preferred for its simplicity, speed, high sensitivity, and resistance to amplification inhibition by the sample. Furthermore, when step (3) is performed by melting curve analysis, a DNA polymerase belonging to Family B that lacks 5' to 3' exonuclease activity is also preferred for use with a fluorescence-quenching probe.

[0046] The conditions for the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80 to 100°C for approximately 0 to 300 seconds (e.g., approximately 0.5 to 300 seconds), and the second and subsequent (repeated) steps (i) may be performed at 80 to 100°C for approximately 0.5 to 300 seconds, step (ii) may be performed at 35 to 80°C for approximately 1 to 300 seconds, and step (iii) may be performed at 35 to 85°C for approximately 1 to 300 seconds. The cycle of steps (i) to (iii) is preferably repeated 30 to 70 times. The temperature and time of the repeated cycles may be changed every 1 to 3 cycles.

[0047] (DNA polymerase) The DNA polymerase that can be used in step (2) is preferably, but is not limited to, a DNA polymerase belonging to Family B. The DNA polymerase belonging to Family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, more preferably a DNA polymerase derived from bacteria of the genera Pyrococcus and Thermococcus. Suitable DNA polymerases also include mutants of Family B archaea that have not lost their DNA polymerase activity. Examples of mutants include those with deletion, substitution, insertion, and / or addition of one to three amino acids in the wild-type amino acid sequence, and those that show 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, DNA polymerase mutants include, but are not limited to, those intended for enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, etc.

[0048] DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants thereof derived therefrom that have not lost their DNA polymerase activity.

[0049] Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9°N-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, as well as mutants thereof that have not lost their DNA polymerase activity. DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3' to 5' exonuclease activity) are particularly suitable for use in the present invention due to their excellent extensibility and thermostability.

[0050] PCR enzymes using these DNA polymerases are commercially available, including Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0051] (KOD-derived DNA polymerase) As used herein, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to a DNA polymerase derived from Thermococcus kodakaraensis and mutants thereof (e.g., a KOD-derived DNA polymerase in which 3' to 5' exonuclease activity has been eliminated by substituting, deleting, inserting, and / or adding one to three amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, step (2) involves performing a nucleic acid amplification reaction using such a KOD-derived DNA polymerase. Compared to Taq DNA polymerase, a DNA polymerase belonging to Family A, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by sample-derived inhibitors. In the present invention, the use of such a KOD DNA polymerase is preferred in terms of simple, rapid, and highly sensitive detection of Mycobacterium mycobacteria, as shown in the Examples below.

[0052] (nucleic acid primer set) The nucleic acid primer set that can be used in step (2) is not particularly limited as long as it can amplify a nucleic acid fragment derived from Mycobacterium acid-fast bacteria that can form a complex with the probe described below. From the viewpoint of easily obtaining more sensitive determination results, the nucleic acid primer set preferably has any one of the following characteristics (G) to (K). (G) a nucleic acid primer set (particularly a nucleic acid primer set for detecting Mycobacterium tuberculosis) capable of amplifying a specific region of SEQ ID NO: 1, preferably a base sequence represented by SEQ ID NO: 2 (particularly bases 50 to 90 of SEQ ID NO: 2); (H) a nucleic acid primer set (particularly a nucleic acid primer set for detecting Mycobacterium avium) capable of amplifying a specific region of SEQ ID NO: 3, preferably a base sequence represented by SEQ ID NO: 4 (particularly, bases 30 to 70 of SEQ ID NO: 4); (I) A nucleic acid primer set (particularly, a nucleic acid primer set for detecting Mycobacterium intracellulare) capable of amplifying a specific region of SEQ ID NO: 5, preferably a nucleotide sequence represented by SEQ ID NO: 6 (particularly, bases 45 to 85 of SEQ ID NO: 6); (J) a nucleic acid primer set (particularly a nucleic acid primer set for detecting Mycobacterium abscessus) capable of amplifying a specific region of SEQ ID NO: 7, preferably a nucleotide sequence represented by SEQ ID NO: 8 (particularly positions 45 to 85 of SEQ ID NO: 8); (K) It is preferable that the nucleic acid primer set is capable of amplifying a specific region of SEQ ID NO: 9, preferably the base sequence shown in SEQ ID NO: 10 (particularly positions 45 to 85 of SEQ ID NO: 10) (particularly a nucleic acid primer set for detecting Mycobacterium kansasii).

[0053] For example, the nucleic acid primer set (G) includes a first primer having a base sequence R1 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence R2 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R1; and a second primer having a base sequence R3 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence R4 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R3, wherein the second primer is complementary to a DNA extension product of the first primer. More preferably, the nucleic acid primer set (G) comprises a first primer having a base sequence S1 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 1 to 65 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S1; and a second primer having a base sequence S3 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 65 to 130 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S3, wherein the second primer is preferably a nucleic acid primer set complementary to a DNA extension product of the first primer.

[0054] More preferably, the nucleic acid primer set (G) comprises a first primer having the nucleotide sequence shown in SEQ ID NO: 12, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, and a second primer having the nucleotide sequence shown in SEQ ID NO: 13, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, wherein one primer is complementary to the DNA extension product of the other primer.

[0055] For example, the nucleic acid primer set (H) includes a first primer having a base sequence R5 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 3 or a base sequence complementary thereto, or a base sequence R6 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R5; and a second primer having a base sequence R7 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 3 or a base sequence complementary thereto, or a base sequence R8 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R7, wherein the second primer is complementary to a DNA extension product of the first primer. More preferably, the nucleic acid primer set (H) comprises a first primer having a base sequence S5 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of positions 1 to 60 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S5; and a second primer having a base sequence S7 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of positions 60 to 120 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S7, wherein the second primer is preferably a nucleic acid primer set complementary to a DNA extension product of the first primer.

[0056] More preferably, the nucleic acid primer set (H) comprises a first primer having the nucleotide sequence shown in SEQ ID NO: 15, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, and a second primer having the nucleotide sequence shown in SEQ ID NO: 16, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, wherein one primer is complementary to the DNA extension product of the other primer.

[0057] For example, the nucleic acid primer set (I) includes a first primer having a base sequence R9 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 5 or a base sequence complementary thereto, or a base sequence R10 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R9; and a second primer having a base sequence R11 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 5 or a base sequence complementary thereto, or a base sequence R12 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R11, wherein the second primer is complementary to a DNA extension product of the first primer. More preferably, the nucleic acid primer set (I) comprises a first primer having a base sequence S9 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 1 to 60 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence S10 in which 1 to 3 (e.g., 1, 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S9; and a second primer having a base sequence S11 of at least 18 consecutive bases (e.g., 18 to 35 bases) in the base sequence from positions 60 to 120 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence S12 in which 1 to 3 (e.g., 1, 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S11, and the second primer is preferably a nucleic acid primer set complementary to a DNA extension product of the first primer.

[0058] More preferably, the nucleic acid primer set (I) comprises a first primer having the nucleotide sequence shown in SEQ ID NO: 18, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in said nucleotide sequence, and a second primer having the nucleotide sequence shown in SEQ ID NO: 19, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in said nucleotide sequence, wherein one primer is complementary to the DNA extension product of the other primer.

[0059] For example, the nucleic acid primer set (J) includes a first primer having a base sequence R13 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 7 or a base sequence complementary thereto, or a base sequence R14 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R13; and a second primer having a base sequence R15 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 7 or a base sequence complementary thereto, or a base sequence R16 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R15, wherein the second primer is complementary to a DNA extension product of the first primer. More preferably, the nucleic acid primer set (J) comprises a first primer having a base sequence S13 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of positions 1 to 60 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S14 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S13; and a second primer having a base sequence S15 of at least 18 consecutive bases (e.g., 18 to 35 bases) in the base sequence of positions 60 to 114 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S16 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S15, and the second primer is preferably a nucleic acid primer set complementary to a DNA extension product of the first primer.

[0060] More preferably, the nucleic acid primer set (J) comprises a first primer having the nucleotide sequence shown in SEQ ID NO: 21, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, and a second primer having the nucleotide sequence shown in SEQ ID NO: 22 or SEQ ID NO: 23, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, wherein one primer is complementary to the DNA extension product of the other primer.

[0061] For example, the nucleic acid primer set (K) includes a first primer having a base sequence R17 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 9 or a complementary base sequence thereof, or a base sequence R18 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R17; and a second primer having a base sequence R19 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of SEQ ID NO: 9 or a complementary base sequence thereof, or a base sequence R20 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence R19, wherein the second primer is complementary to a DNA extension product of the first primer. More preferably, the nucleic acid primer set (K) comprises a first primer having a base sequence S17 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence from positions 1 to 65 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence S18 in which 1 to 3 (e.g., 1, 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S17; and a second primer having a base sequence S19 of at least 18 consecutive bases (e.g., 18 to 35 bases) in the base sequence from positions 65 to 130 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence S20 in which 1 to 3 (e.g., 1, 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence S19, and the second primer is preferably a nucleic acid primer set complementary to a DNA extension product of the first primer.

[0062] More preferably, the nucleic acid primer set (K) comprises a first primer having the nucleotide sequence shown in SEQ ID NO: 25, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, and a second primer having the nucleotide sequence shown in SEQ ID NO: 26, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted, or added in said nucleotide sequence, wherein one primer is complementary to the DNA extension product of the other primer.

[0063] Regarding the base sequence of the primer, substitution, deletion, insertion, and addition may be collectively referred to as "mutation." A preferred mutation is substitution. Bases substituted from the original base sequence may include adenine, thymine, cytosine, guanine, as well as uracil, universal bases, and mixed bases. The degree of mutation is not particularly limited as long as it can hybridize with part or all of the nucleic acid derived from Mycobacterium acid-fast bacteria to form a complex.

[0064] The term "universal base" as used herein refers to a base that can form a base pair with any of the four nucleic acid bases, adenine, cytosine, guanine, and thymine, or that does not form a base pair with any of the four nucleic acid bases. Any base that has the above properties can be used as a universal base, but deoxyinosine and 5-nitroindole are preferred.

[0065] The term "mixed base" as used herein refers to a plurality of bases at a specific site constituting an oligonucleotide. Mixed bases are designated by a single letter of the alphabet by IUPAC, with A or T being represented as W, A or G as R, A or C as M, T or G as K, T or C as Y, G or C as S, A or C or T as H, G or C or T as B, A or G or C as V, A or G or T as D, and if any of A, T, G, and C are possible, then they are represented as N. This specification will also follow this notation.

[0066] As shown in the results of the Examples described below, it has been confirmed that Mycobacterium acid-fast bacteria can be detected with sufficient sensitivity even when primers designed in different regions with a shift of 1 to 3 bases are used.

[0067] The primers can be used in any nucleic acid amplification method, but are preferably PCR primers used in PCR, particularly those used as part of a PCR primer set designed so that one primer is complementary to the nucleic acid extension product of the other primer to enable a PCR reaction.

[0068] [Process (3)] Step (3) may be performed by any method known in the art. Mycobacterium spp., the target Mycobacterium spp., may mutate, similar to other infectious microorganisms. Mismatches between the base sequence of a primer or probe and the base sequence of a target Mycobacterium spp. mutant may reduce the binding strength of the primer or probe to the target gene or its derived nucleic acid amplification product (target nucleic acid). In particular, in real-time PCR, if there are many mismatches between the target nucleic acid and the probe, the probe may not be able to bind sufficiently to the target nucleic acid, resulting in a delayed or complete absence of an amplification curve rise, which may lead to false negatives. In melting curve analysis, step (3) is performed after PCR is completed. Therefore, even if there are mismatches in the primers or probe, detection is possible as long as the final nucleic acid amplification product is obtained. Therefore, the effect of mismatches is less significant than in real-time PCR. Therefore, in step (3), it is particularly preferable to detect the nucleic acid amplification product using melting curve analysis. Furthermore, by detecting nucleic acid amplification products using melting curve analysis, it is possible to detect them in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less from the start of the nucleic acid amplification reaction to the completion of melting curve analysis).

[0069] In one embodiment, the step (3) preferably includes the following steps (3-1) and (3-2): (3-1) hybridizing one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) with one or more nucleic acid probes of the present invention to form a complex; and (3-2) A step of detecting the complex of step (3-1). In order to obtain highly sensitive determination results in the detection of Mycobacterium mycobacteria, it is preferable to use the nucleic acid probe described below, which can specifically react with the nucleic acid amplification product that can be produced in the nucleic acid amplification reaction of step (2) to form a complex.

[0070] The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of the nucleic acid amplification product with the nucleic acid probe, such as, but not limited to, a temperature that is at least 5°C lower, more preferably at least 10°C lower than the Tm value of the nucleic acid probe.

[0071] (nucleic acid probe) The nucleic acid probe of the present invention preferably has any one of the characteristics (A) to (E) and the characteristic (F). When the nucleic acid probe is labeled with a fluorescence quenching dye that quenches fluorescence by interaction with guanine, as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0072] In one embodiment, the nucleic acid probe of the present invention can detect at least one of Mycobacterium tuberculosis (including Mycobacterium tuberculosis mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 1, Mycobacterium avium (including Mycobacterium avium mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 3, Mycobacterium intracellulare (including Mycobacterium intracellulare mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 5, Mycobacterium abscessus (including Mycobacterium abscessus mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 7, and Mycobacterium kansasii (including Mycobacterium kansasii mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 9.

[0073] Preferably, the nucleic acid probe of the present invention can detect at least one of Mycobacterium tuberculosis (including Mycobacterium tuberculosis mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 2, Mycobacterium avium (including Mycobacterium avium mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 4, Mycobacterium intracellulare (including Mycobacterium intracellulare mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 6, Mycobacterium abscessus (including Mycobacterium abscessus mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 8, and Mycobacterium kansasii (including Mycobacterium kansasii mutants) having a nucleotide sequence that is 85% or more identical to the nucleotide sequence of SEQ ID NO: 10.

[0074] More preferably, the nucleic acid probe of the present invention is directed to Mycobacterium tuberculosis (including mutants of Mycobacterium tuberculosis) having a nucleotide sequence showing 85% or more identity with the nucleotide sequence of positions 50 to 90 of SEQ ID NO: 2, Mycobacterium avium (including mutants of Mycobacterium avium) having a nucleotide sequence showing 85% or more identity with the nucleotide sequence of positions 30 to 70 of SEQ ID NO: 4, or Mycobacterium avium (including mutants of Mycobacterium avium) having a nucleotide sequence showing 85% or more identity with the nucleotide sequence of positions 45 to 85 of SEQ ID NO: 6. At least one of Mycobacterium intracellulare (including Mycobacterium intracellulare mutants), Mycobacterium abscessus (including Mycobacterium abscessus mutants) having a base sequence that is 85% or more identical to the base sequence of positions 45 to 85 of SEQ ID NO: 8, and Mycobacterium kansasii (including Mycobacterium kansasii mutants) having a base sequence that is 85% or more identical to the base sequence of positions 45 to 85 of SEQ ID NO: 10 can be detected.

[0075] Furthermore, the nucleic acid probe of the present invention is suitable for Mycobacterium tuberculosis (including mutants of Mycobacterium tuberculosis) having a nucleotide sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity with the nucleotide sequence of bases 50 to 90 of SEQ ID NO: 2; Mycobacterium avium (including mutants of Mycobacterium avium) having a nucleotide sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity with the nucleotide sequence of bases 30 to 70 of SEQ ID NO: 4; and Mycobacterium avium (including mutants of Mycobacterium avium) having a nucleotide sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity with the nucleotide sequence of bases 45 to 85 of SEQ ID NO: 6. the nucleic acid probe can be capable of detecting at least one of Mycobacterium intracellulare (including Mycobacterium intracellulare mutants) having a base sequence shown in SEQ ID NO: 8; Mycobacterium abscessus (including Mycobacterium abscessus mutants) having a base sequence that is 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identical to the base sequence of bases 45 to 85 of SEQ ID NO: 8; and Mycobacterium kansasii (including Mycobacterium kansasii mutants) having a base sequence that is 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identical to the base sequence of bases 45 to 85 of SEQ ID NO: 10.

[0076] In a specific embodiment, the nucleic acid probe of the present invention may be a probe containing a base sequence represented by A1, B1, C1, D1, or E1 in which 1 to 3 bases have been substituted, deleted, inserted, or added. The number of bases that may be substituted, deleted, inserted, or added may preferably be 1 or 2. When a probe contains such a base substitution, deletion, insertion, or addition, it is said to contain a mismatched base, and it has been confirmed in the Examples described below that probes containing such mismatched bases can also be suitably used in the present invention.

[0077] As used herein, "containing a mismatched base" (or simply "mismatch") means containing a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid becomes double-stranded after a nucleic acid amplification reaction, the base sequence of one of the single-stranded nucleic acids resulting from the dissociation of the double strand). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., an adenine base, a cytosine base, a thymine base, or a universal base). For example, when detecting a region of a target nucleic acid that is susceptible to mutation, a mismatched base (e.g., a universal base) can be selected for the position of the probe corresponding to the susceptible base.

[0078] When the nucleic acid probe of the present invention contains a mismatched base, the position of the mismatched base is not particularly limited as long as it does not inhibit the effects of the present invention. From the viewpoint of more reliably detecting the nucleic acid amplification product, it is preferable that the mismatched base is not a terminal base of each probe. For example, the position of the mismatched base is preferably within 5 mers before and after the center of the total length n of the base sequence constituting the probe ((n+1) / 2 when n is odd, n / 2 when n is even), more preferably within 4 mers before and after, even more preferably within 3 mers before and after, even more preferably within 2 mers before and after, and particularly preferably within 1 mer before and after.

[0079] The nucleic acid probe for detecting Mycobacterium tuberculosis (including mutants of Mycobacterium tuberculosis) of the present invention is preferably a fluorescently labeled probe having the base sequence shown in SEQ ID NO: 11 or its complementary base sequence as the base sequence of (A).

[0080] The nucleic acid probe for detecting Mycobacterium avium (including mutants of Mycobacterium avium) of the present invention is preferably a fluorescently labeled probe having the base sequence (B) shown in any one of SEQ ID NOs: 14, 33, 34, 35, and 36 or a complementary base sequence thereto.

[0081] The nucleic acid probe for detecting Mycobacterium intracellulare (including mutants of Mycobacterium intracellulare) of the present invention is preferably a fluorescently labeled probe having the base sequence (C) shown in any one of SEQ ID NOs: 17, 37, 38, 39, 40, 41, and 42 or a complementary base sequence thereof.

[0082] The nucleic acid probe for detecting Mycobacterium abscessus (including mutants of Mycobacterium abscessus) of the present invention is preferably a fluorescently labeled probe having the base sequence shown in SEQ ID NO: 20 as the base sequence of (D) or a complementary base sequence thereto.

[0083] The nucleic acid probe for detecting Mycobacterium kansasii (including mutants of Mycobacterium kansasii) of the present invention is preferably a fluorescently labeled probe having the base sequence shown in SEQ ID NO: 24 or its complementary base sequence as the base sequence of (E).

[0084] The length of the nucleic acid probe of the present invention (particularly the base sequences (A) to (E)) is not particularly limited as long as it is 10 bases or more, and is, for example, 11 bases or more, preferably 12 bases or more, and is usually 25 bases or less, preferably 22 bases or less, more preferably 20 bases or less, 18 bases or less, or 17 bases or less. The length of the nucleic acid probe of the present invention is more preferably 10 to 25 bases, even more preferably 11 to 20 bases, and even more preferably 12 to 20 bases. In a specific embodiment, the length of the nucleic acid probe of the present invention may be 12 to 18 bases, 12 to 17 bases, or 14 to 20 bases. By using a probe of such a length, Mycobacterium species can be detected with higher sensitivity.

[0085] In a particularly preferred embodiment, specific examples of the nucleic acid probe of the present invention include probes containing the nucleotide sequence set forth in any one of SEQ ID NOs: 11, 14, 33, 34, 35, 36, 17, 37, 38, 39, 40, 41, 42, 20, and 24, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides have been substituted, deleted, inserted, or added in these nucleotide sequences. By using a probe having such a specific nucleotide sequence, Mycobacterium species can be detected with even higher sensitivity.

[0086] The above-mentioned probe is preferably labeled at only either the 5'-end or the 3'-end. In one embodiment, the nucleic acid probe of the present invention is preferably labeled so as to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 85% or more, preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity to a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably is labeled so as to generate quenching. The labeling substance is not particularly limited, but is more preferably a fluorescent dye.

[0087] The fluorescent dye may be either a fluorescent substance that emits fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that quenches fluorescence by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that quenches fluorescence by interaction with guanine). Specific examples include, but are not limited to, at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665).

[0088] More specifically, examples of fluorescence quenching dyes that are quenched by interaction with guanine include at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G (CR6G), TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

[0089] In the present invention, it is preferable to design a probe so that the terminal base labeled with a fluorescence quenching dye is cytosine. When such a probe hybridizes to a nucleic acid amplification product, it forms a base pair with a guanine base in the nucleic acid amplification product, thereby quenching the fluorescence, and therefore, it is possible to measure the change in the fluorescence intensity of the reaction solution very easily.

[0090] When the probe hybridizes, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and this technology can also be referenced in the present invention. That is, when the probe hybridizes, quenching can be achieved if the guanine base in the nucleic acid amplification product is located within, for example, 1 to 3 bases of the cytosine base of the probe (the base that forms a base pair with the cytosine base is counted as 1).

[0091] Therefore, even if at least one terminal base labeled with a fluorescence quenching dye is not cytosine, the change in fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5354216, and this technology can also be used in the present invention. For example, when the probe hybridizes, quenching can be achieved if a guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases of the terminal base labeled with the fluorescence quenching dye (the base that forms a base pair with the terminal base is counted as 1).

[0092] In a particularly preferred embodiment, the nucleic acid probe of the present invention is used in step (3). In this manner, Mycobacterium species can be detected using the nucleic acid probe of the present invention. The method of the present invention may use one type of probe of the present invention or a combination of two or more types of probes of the present invention.

[0093] In one embodiment, step (3) may include at least one of the following steps (3-a), (3-b), and (3-c): (3-a) Simultaneously with step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution to monitor the progress of the reaction (nucleic acid amplification reaction) in step (2) in real time. (3-b) After completion of step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution to monitor the progress of the reaction (nucleic acid amplification reaction) in step (2) at an endpoint. (3-c) After step (2), hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution.

[0094] Step (3-a), (3-b), or (3-c) allows for simple, rapid, and highly sensitive detection of the formation of a complex between a nucleic acid amplification product and a nucleic acid probe. Steps (3-a), (3-b), and (3-c) may be performed in combination; for example, both steps (3-a) and (3-b) or both steps (3-a) and (3-c) may be performed. In one embodiment, step (3-b) or (3-c) is preferred from the viewpoint of more rapid detection of a nucleic acid amplification product. Step (3-c), i.e., melting curve analysis, is particularly preferred.

[0095] (Step (3-a)) Step (3-a) is a method for monitoring the progress of the nucleic acid amplification reaction in real time (so-called real-time PCR method), and quantitative analysis is possible by comparing with a control substance of known concentration.

[0096] (Step (3-b)) In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the endpoint, allowing rapid detection of the target nucleic acid contained in the sample. Furthermore, the approximate amount of target nucleic acid can be estimated by comparing the fluorescence intensity at the endpoint. For example, the progress of a nucleic acid amplification reaction is monitored at an endpoint by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescence quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured and compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming whether or not the target nucleic acid has been amplified. Alternatively, the presence or absence of the target nucleic acid in a sample can be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. A control reaction solution is a reaction solution to which a sample known to be negative or positive has been added instead of the sample to be measured. The progress of the nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of more rapid and simple detection, it is preferable to measure at the end point.

[0097] (Step (3-c)) In step (3-c), measuring the temperature dependence of fluorescence intensity can specifically mean measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the nucleic acid probe used can be determined by first differentiating the obtained fluorescence intensity with respect to temperature. Furthermore, the fluorescence intensity may be converted into a fluorescence quenching rate or the like depending on the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. Generally, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that remains single-stranded. Because the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms in target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0098] For example, melting curve analysis is also used in SNP analysis. If there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases will mismatch when the probe hybridizes, and the Tm value will generally be low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm value.

[0099] [Reagents for detecting Mycobacterium species] In another embodiment, the present invention provides a reagent for detecting Mycobacterium species. The reagent preferably contains at least components necessary for nucleic acid amplification and detection, in addition to the nucleic acid probe or probe set of the present invention described above. These necessary components may be publicly known. For example, the reagent of the present invention preferably contains at least a PCR nucleic acid primer set, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and an inorganic salt such as a magnesium salt. The nucleic acid primer set for PCR and the nucleic acid probe for detection may include a plurality of sets for amplifying a plurality of regions of Mycobacterium mycobacteria species, and preferably includes at least one of a probe for detecting Mycobacterium tuberculosis having the characteristics (A) and (F) above, a probe for detecting Mycobacterium avium having the characteristics (B) and (F) above, a probe for detecting Mycobacterium intracellulare having the characteristics (C) and (F) above, a probe for detecting Mycobacterium abscessus having the characteristics (D) and (F) above, and a probe for detecting Mycobacterium kansasii having the characteristics (E) and (F) above, and the regions to which each of these probes can bind are included. The nucleic acid probe may comprise a primer (or primer set) capable of amplifying a region of interest, more preferably a combination of at least one of a primer (or primer set) for detecting Mycobacterium tuberculosis having the characteristic (G), a primer (or primer set) for detecting Mycobacterium avium having the characteristic (H), a primer (or primer set) for detecting Mycobacterium intracellulare having the characteristic (I), a primer (or primer set) for detecting Mycobacterium abscessus having the characteristic (J), and a primer (or primer set) for detecting Mycobacterium kansasii having the characteristic (K). The concentration of each component can be adjusted as appropriate; for example, the nucleic acid probe is preferably 0.01 to 1 μM, more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, it is preferable that each nucleic acid probe contained in the probe set is within the above-mentioned concentration range.The nucleic acid primer is preferably 0.01 to 10 μM. The DNA polymerase is preferably 0.01 to 1 U / μL, more preferably 0.02 to 0.8 U / μL. The deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The inorganic salts such as magnesium salts are preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0100] Furthermore, the reagent of the present invention may contain additives known in the art for the purposes of suppressing nonspecific amplification or promoting the reaction. Examples of additives for suppressing nonspecific amplification include known anti-DNA polymerase antibodies and phosphate. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, and Nonidet® P40. In addition, to facilitate the determination of false negatives, the reagent of the present invention preferably contains an internal control known in the art. In the present invention, these additives may be used alone or in combination of two or more.

[0101] In certain embodiments, the reagent of the present invention preferably includes an internal control. Using an internal control in combination makes it easy to confirm that the nucleic acid amplification reaction is proceeding normally, reduces the risk of false negatives, and enables more accurate testing for Mycobacterium mycobacteria. When using an internal control in combination in melting curve analysis, the detection temperatures of the internal control and the probe for detecting Mycobacterium mycobacteria of the present invention are preferably different by a certain degree (e.g., 5°C or more). For example, when using an internal control and / or the probe for detecting Mycobacterium mycobacteria of the present invention in melting curve analysis, the detection temperatures can be determined by methods known in the art, and those skilled in the art can appropriately select and use those whose detection temperatures differ from each other by, for example, 5°C or more.

[0102] [Kit for detecting Mycobacterium mycobacteria] In another embodiment, the present invention provides a kit for detecting Mycobacterium species. The kit of the present invention is not particularly limited as long as it contains the nucleic acid probe or probe set of the present invention described above, and, if necessary, further contains at least a reagent of the present invention containing the primers (or primer set) described above, and is configured to detect Mycobacterium species (including differentiation of Mycobacterium species). For example, the kit of the present invention can optionally contain a reagent capable of detecting or quantifying the presence of the target Mycobacterium species and / or instructions for use. For example, the kit of the present invention can be provided in a form in which the nucleic acid probe or probe set, components necessary for the nucleic acid amplification reaction, and components necessary for detecting the amplification product are sealed in the same container or in separate containers, packaged in a single package, and includes information on how to use the kit. The kit of the present invention can also contain a positive control solution and / or a negative control solution. [Example]

[0103] The present invention will be specifically described below based on test examples, but the present invention is not limited to the following test examples.

[0104] Test Example 1: Evaluation of multicopy number of target genes in Mycobacterium To evaluate the copy number of the target gene in M. tuberculosis, the RVBD_0094c gene (SEQ ID NO: 1) of the Mycobacterium tuberculosis H37Rv strain, registered in GenBank (National Center for Biotechnology Information), was searched using the Basic Local Alignment Search Tool (Blast). Genes with 80% or more partial identity to the RVBD_0094c gene are listed in Table 1. Since there are three genes with 80% or more identity in the genome, it is estimated that the RVBD_0094c gene has three copies. Genbank data indicates that the RVBD_0094c gene encodes a hypothetical protein (Protein ID: AFN47941.1), which is composed of a protein consisting of a DUF222 domain and a HNH nuclease domain. To evaluate the copy number of the target gene in Mycobacterium avium, we used the Basic Local Alignment Search Tool (Blast) to search for the MAA44156_02837 gene (SEQ ID NO: 3) of M. avium subsp. avium strain DSM 44156, which is registered in GenBank (National Center for Biotechnology Information). Genes with 80% or more partial identity to the MAA44156_02837 gene are listed in Table 2. Since there are five genes with 80% or more identity in the genome, the MAA44156_02837 gene is estimated to have five copies. Genbank data indicates that the MAA44156_02837 gene encodes a hypothetical protein (Protein ID: QGW33033.1), which is composed of a protein consisting of a DUF222 domain and a HNH nuclease domain. To evaluate the copy number of the target gene in Mycobacterium intracellulare, we performed a search using the Basic Local Alignment Search Tool (Blast) for the KN251_15665 gene (SEQ ID NO: 5) of the M. intracellulare ATCC 13950 strain, registered with GenBank (National Center for Biotechnology Information). Table 3 lists genes with 80% or more partial identity to the KN251_15665 gene. Since there are four genes with 80% or more identity in the genome, the KN251_15665 gene is estimated to have four copies. Genbank data indicates that the KN251_15665 gene encodes an HNH endonuclease signature motif-containing protein (Protein ID: UQC05651.2). To evaluate the copy number of the target gene in M. abscessus, we used the Basic Local Alignment Search Tool (Blast) to search for the MAB_0990c gene (SEQ ID NO: 7) of the M. abscessus ATCC 19977 strain, registered with GenBank (National Center for Biotechnology Information). Genes with 60% or greater partial identity to the MAB_0990c gene are listed in Table 4. Genbank data indicates that the MAB_0990c gene encodes a conserved hypothetical protein (Protein ID: CAM61082.1), which is composed of a protein consisting of a DUF222 domain and an HNH nuclease domain. In M. abscessus, a fragmented sequence similar to a portion of the MAB_0990c gene is present in other sequences. Since there are five fragments with 80% or greater identity in the genome, it is reasonable to consider that there are five copies of a portion of the MAB_0990c gene. To evaluate the copy number of the target gene in M. kansasii, the MKAN_05295 gene (SEQ ID NO: 9) of the M. kansasii ATCC 12478 strain, registered with GenBank (National Center for Biotechnology Information), was searched using the Basic Local Alignment Search Tool (Blast). Genes with 80% or more partial identity to the MKAN_05295 gene are listed in Table 5. Since there are three genes with 80% or more identity in the genome, it is estimated that the MKAN_05295 gene has three copies. Genbank data indicates that the MKAN_05295 gene encodes a hypothetical protein (Protein ID: AGZ49763.1), which is composed of a protein consisting of a DUF222 domain and a HNH nuclease domain.

[0105] [Table 1]

[0106] [Table 2]

[0107] [Table 3]

[0108] [Table 4]

[0109] [Table 5]

[0110] Test Example 2: Evaluation of cross-reactivity of target genes in Mycobacterium The RVBD_0094c gene (SEQ ID NO: 1) of the Mycobacterium tuberculosis H37Rv strain, a target gene for Mycobacterium tuberculosis, was evaluated for cross-reactivity with other mycobacteria using Blast (Basic Local Alignment Search Tool). The percent identity (%) of each mycobacterial species to SEQ ID NO: 1 is shown in Table 6. These results demonstrate that the RVBD_0094c gene of the Mycobacterium tuberculosis H37Rv strain is highly identical only among Mycobacterium tuberculosis strains and has low identity with other mycobacteria, making it unlikely to cross-react and highly specific. The MAA44156_02837 gene (SEQ ID NO: 3) of M. avium subsp. avium strain DSM 44156, a target gene in Mycobacterium avium, was evaluated for cross-reactivity with other mycobacteria using Blast (Basic Local Alignment Search Tool). The percent identity (%) of SEQ ID NO: 3 among various mycobacteria is shown in Table 7. These results demonstrate that the MAA44156_02837 gene of M. avium subsp. avium strain DSM 44156 is highly identical only among Mycobacterium avium strains and has low identity with other mycobacteria, making it unlikely to cross-react and highly specific. The KN251_15665 gene (SEQ ID NO: 5) of the M. intracellulare ATCC 13950 strain, a target gene in Mycobacterium intracellulare, was evaluated for cross-reactivity with other mycobacteria using Blast (Basic Local Alignment Search Tool). The percent identity (%) of SEQ ID NO: 5 among various mycobacteria is shown in Table 8. These results demonstrate that the KN251_15665 gene of the M. intracellulare ATCC 13950 strain is highly identical only among Mycobacterium intracellulare strains and has low identity with other mycobacteria, making it unlikely to cross-react and highly specific. The target gene for Mycobacterium abscessus, the MAB_0990c gene of M. abscessus ATCC 19977, is a multicopy gene with a portion fragmented. Therefore, the partial sequence of the MAB_0990c gene, SEQ ID NO: 8, was evaluated for cross-reactivity with other mycobacteria using Blast (Basic Local Alignment Search Tool). The percent identity of each mycobacterial species to SEQ ID NO: 8 is shown in Table 9. These results demonstrate that the MAB_0990c gene of M. abscessus ATCC 19977 is highly identical only among Mycobacterium abscessus strains and has low identity with other mycobacteria, making it unlikely to cross-react and highly specific. The MKAN_05295 gene (SEQ ID NO: 9) of the M. kansasii ATCC 12478 target gene was evaluated for cross-reactivity with other mycobacteria using Blast (Basic Local Alignment Search Tool). The percent identity (%) of SEQ ID NO: 9 among various mycobacteria is shown in Table 10. These results demonstrate that the MKAN_05295 gene of the M. kansasii ATCC 12478 strain is highly identical only among Mycobacterium kansasii strains and has low identity with other mycobacteria, making it unlikely to cross-react and highly specific.

[0111] [Table 6]

[0112] [Table 7]

[0113] [Table 8]

[0114] [Table 9]

[0115] [Table 10]

[0116] Test Example 3: Evaluation of differences between strains of the same species in target genes of Mycobacterium spp. The RVBD_0094c gene (SEQ ID NO: 1) of the Mycobacterium tuberculosis H37Rv target gene was examined for the presence of each Mycobacterium tuberculosis strain registered on Blast (Basic Local Alignment Search Tool). For comparison, the single-copy gtf gene (Non-Patent Document 5) and the multi-copy IS6110 gene (Non-Patent Document 3), which are widely used in genetic testing for Mycobacterium tuberculosis, were selected. The number of Mycobacterium tuberculosis strains that were found by searching these gene sequences using Blast is shown in Table 11. The gtf gene was present in 93% of strains, and the IS6110 gene was present in 98% of strains. However, the RVBD_0094c gene was present in 100% of strains, demonstrating that no strains were deficient. These results suggest that the RVBD_0094c gene is the only gene that can improve detection sensitivity through multicopy and prevent missed detections due to differences between strains when conducting genetic testing for Mycobacterium tuberculosis. The target gene in Mycobacterium avium, the MAA44156_02837 gene (SEQ ID NO: 3) of M. avium subsp. avium strain DSM 44156, was examined for the presence of this gene in each Mycobacterium avium strain registered on Blast (Basic Local Alignment Search Tool). For comparison, the single-copy hsp70 gene (Non-Patent Document 5) and the multi-copy IS1245 and IS1311 genes (Non-Patent Document 4), which are widely used in genetic testing of Mycobacterium avium, were selected. The number of Mycobacterium avium strains that were found by searching these gene sequences using Blast is shown in Table 12. The hsp70 gene was present in 100%, the IS1245 gene in 50%, and the IS1311 gene in 98% of the strains, while the MAA44156_02837 gene was present in 100% of the strains, with no deletions. These results suggest that when conducting genetic testing for Mycobacterium avium, the MAA44156_02837 gene is the only gene that can improve detection sensitivity through multicopy and prevent missed detections due to differences between strains. The target gene in Mycobacterium intracellulare, the KN251_15665 gene (SEQ ID NO: 5) of the M. intracellulare ATCC 13950 strain, was examined for the presence of this gene in each Mycobacterium intracellulare strain registered on Blast (Basic Local Alignment Search Tool). The nor gene, a single-copy gene (Non-Patent Document 5), was selected for comparison. Furthermore, no multi-copy genes capable of detecting Mycobacterium intracellulare have been known to date. The number of Mycobacterium intracellulare strains that were found by searching these gene sequences with Blast is shown in Table 13. While the nor gene was present in 100% of the strains, the KN251_15665 gene was present in 100% of the strains, and no strains were found to be deficient. These results suggest that when conducting genetic testing for Mycobacterium intracellulare, the KN251_15665 gene is the only gene that can improve detection sensitivity through multicopy and prevent missed detections due to differences between strains. The target gene in Mycobacterium abscessus, the MAB_0990c gene (SEQ ID NO: 7) of the M. abscessus ATCC 19977 strain, was examined for the presence of this gene in each Mycobacterium abscessus strain registered on Blast (Basic Local Alignment Search Tool). The single-copy erm gene (Non-Patent Document 5) was selected for comparison. Furthermore, no multi-copy genes capable of detecting Mycobacterium abscessus have been known to date. The number of Mycobacterium abscessus strains found in a Blast search of these gene sequences is shown in Table 14. While the erm gene was present in 100% of the strains, the MAB_0990c gene was present in 100% of the strains, with no deletions. These results suggest that when conducting genetic testing for Mycobacterium abscessus, the MAB_0990c gene is the only gene that can improve detection sensitivity through multicopy and prevent missed detections due to differences between strains. The MKAN_05295 gene (SEQ ID NO: 9) of the M. kansasii ATCC 12478 target gene was examined for the presence of each Mycobacterium kansasii strain registered on Blast (Basic Local Alignment Search Tool). The single-copy ITS gene (Non-Patent Document 5) was selected for comparison. Furthermore, no multi-copy genes capable of detecting Mycobacterium kansasii have been known to date. The number of Mycobacterium kansasii strains that were found by searching these gene sequences with Blast is shown in Table 15. While the ITS gene was present in 100% of the strains, the MKAN_05295 gene was present in 100% of the strains, and no strains were found to be deficient. These results suggest that when conducting genetic testing for Mycobacterium kansasii, the MKAN_05295 gene is the only gene that can improve detection sensitivity through multicopy and prevent missed detections due to differences between strains.

[0117] [Table 11]

[0118] [Table 12]

[0119] [Table 13]

[0120] [Table 14]

[0121] [Table 15]

[0122] Test Example 4: Detection of Mycobacterium tuberculosis (4-1) Method Mycobacterium tuberculosis DNA (Vircell, AMPLIRUN® MYCOBACTERIUM TUBERCULOSIS DNA CONTROL), Mycobacterium avium DNA (Vircell, AMPLIRUN® MYCOBACTERIUM AVIUM DNA CONTROL), Mycobacterium intracellulare DNA (Vircell, AMPLIRUN® MYCOBACTERIUM INTRACELLULARE DNA CONTROL), Mycobacterium abscessus DNA (ATCC, Genomic DNA from Mycobacterium abscessus strain L948 (ATCC19977)), Mycobacterium kansasii DNA (Vircell, AMPLIRUN® MYCOBACTERIUM KANSASII DNA) The specific detection of Mycobacterium tuberculosis DNA was confirmed using 100 or 20 copies of the control per reaction. Purified water was used as a negative sample to check for nonspecific amplification. The primer set for detecting Mycobacterium tuberculosis consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 12 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 13. The probe for detecting Mycobacterium tuberculosis was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11, synthesized according to standard methods (labeled at the 3' end with BODIPY-FL; see Figure 6 for the labeling position on the probe). Measurements were performed in duplicate, and the average rate of fluorescence change was calculated.

[0123] (4-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 0.5 μM primer consisting of the base sequence shown in SEQ ID NO: 12 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 13 0.5 μM of the probe shown in SEQ ID NO: 11 (labeled at the 3' end with BODIPY-FL)

[0124] (4-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec) The measurement time in this example was approximately 30 minutes, enabling rapid detection of Mycobacterium tuberculosis DNA.

[0125] (4-4) Results Figure 1 shows a detection graph obtained when 100 copies of Mycobacterium tuberculosis DNA were detected by nucleic acid amplification and melting curve analysis. The measurement results for the primers and probes used in this test example are summarized in Table 16. The rate of change in fluorescence (represented as the quenching rate in this example) was calculated using the formula: (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0126] As shown in the results in Table 16, it was confirmed that the primers and probes used in the test example specifically detected only Mycobacterium tuberculosis DNA, and did not detect Mycobacterium avium DNA, Mycobacterium intracellulare DNA, Mycobacterium abscessus DNA, or Mycobacterium kansasii DNA. In addition, when purified water was used as a negative sample to check for non-specific amplification, no non-specific amplification was observed.

[0127] [Table 16]

[0128] Test Example 5: Detection of Mycobacterium avium (5-1) Method Mycobacterium tuberculosis DNA (Vircell, AMPLIRUN® MYCOBACTERIUM TUBERCULOSIS DNA CONTROL), Mycobacterium avium DNA (Vircell, AMPLIRUN® MYCOBACTERIUM AVIUM DNA CONTROL), Mycobacterium intracellulare DNA (Vircell, AMPLIRUN® MYCOBACTERIUM INTRACELLULARE DNA CONTROL), Mycobacterium abscessus DNA (ATCC, Genomic DNA from Mycobacterium abscessus strain L948 (ATCC19977)), Mycobacterium kansasii DNA (Vircell, AMPLIRUN® MYCOBACTERIUM KANSASII DNA) The specific detection of Mycobacterium avium DNA was confirmed using 100 or 20 copies of the control per reaction. Purified water was used as a negative sample to check for nonspecific amplification. The Mycobacterium avium detection primer set consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 15 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 16. The Mycobacterium avium detection probe was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, synthesized according to standard methods (labeled at the 3' end with BODIPY-FL; see Figure 7 for the labeling position on the probe). Measurements were performed in duplicate, and the average rate of fluorescence change was calculated.

[0129] (5-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 0.5 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 16 0.5 μM of the probe shown in SEQ ID NO: 14 (labeled at the 3' end with BODIPY-FL)

[0130] (5-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec) The measurement time in this example was approximately 30 minutes, enabling rapid detection of Mycobacterium avium DNA.

[0131] (5-4) Results Figure 2 shows a detection graph obtained when 100 copies of Mycobacterium avium DNA were detected by nucleic acid amplification and melting curve analysis. The measurement results for the primers and probes used in this test example are summarized in Table 17. The fluorescence change rate (represented as the quenching rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0132] As shown in the results in Table 17, it was confirmed that the primers and probes used in the test example specifically detected only Mycobacterium avium DNA, and did not detect Mycobacterium tuberculosis DNA, Mycobacterium intracellulare DNA, Mycobacterium abscessus DNA, or Mycobacterium kansasii DNA. In addition, when purified water was used as a negative sample to check for non-specific amplification, no non-specific amplification was observed.

[0133] [Table 17]

[0134] Test Example 6: Detection of Mycobacterium intracellulare (6-1) Method Mycobacterium tuberculosis DNA (Vircell, AMPLIRUN® MYCOBACTERIUM TUBERCULOSIS DNA CONTROL), Mycobacterium avium DNA (Vircell, AMPLIRUN® MYCOBACTERIUM AVIUM DNA CONTROL), Mycobacterium intracellulare DNA (Vircell, AMPLIRUN® MYCOBACTERIUM INTRACELLULARE DNA CONTROL), Mycobacterium abscessus DNA (ATCC, Genomic DNA from Mycobacterium abscessus strain L948 (ATCC19977)), Mycobacterium kansasii DNA (Vircell, AMPLIRUN® MYCOBACTERIUM KANSASII DNA) The specific detection of Mycobacterium intracellulare DNA was confirmed using 100 or 20 copies of the control per reaction. Purified water was used as a negative sample to check for nonspecific amplification. The primer set for Mycobacterium intracellulare detection consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 18 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 19. The probe for Mycobacterium intracellulare detection was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, synthesized according to standard methods (labeled at the 5' end with CR6G and phosphorylated at the 3' end; see Figure 8 for the labeling position on the probe). Measurements were performed in duplicate, and the average rate of fluorescence change was calculated.

[0135] (6-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 18 0.5 μM primer consisting of the base sequence shown in SEQ ID NO: 19 0.5 μM of the probe shown in SEQ ID NO: 17 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0136] (6-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec) The measurement time in this example was approximately 30 minutes, enabling rapid detection of Mycobacterium intracellulare DNA.

[0137] (6-4)Result Figure 3 shows a detection graph obtained when 100 copies of Mycobacterium intracellulare DNA were detected by nucleic acid amplification and melting curve analysis. The measurement results for the primers and probes used in this test example are summarized in Table 18. The rate of change in fluorescence (represented as the quenching rate in this example) was calculated using the formula: (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0138] As shown in the results in Table 18, it was confirmed that the primers and probes used in the test example specifically detected only Mycobacterium intracellulare DNA, and did not detect Mycobacterium tuberculosis DNA, Mycobacterium avium DNA, Mycobacterium abscessus DNA, or Mycobacterium kansasii DNA. In addition, when purified water was used as a negative sample to check for non-specific amplification, no non-specific amplification was observed.

[0139] [Table 18]

[0140] Test Example 7: Detection of Mycobacterium abscessus (7-1) Method Mycobacterium tuberculosis DNA (Vircell, AMPLIRUN® MYCOBACTERIUM TUBERCULOSIS DNA CONTROL), Mycobacterium avium DNA (Vircell, AMPLIRUN® MYCOBACTERIUM AVIUM DNA CONTROL), Mycobacterium intracellulare DNA (Vircell, AMPLIRUN® MYCOBACTERIUM INTRACELLULARE DNA CONTROL), Mycobacterium abscessus DNA (ATCC, Genomic DNA from Mycobacterium abscessus strain L948 (ATCC19977)), Mycobacterium kansasii DNA (Vircell, AMPLIRUN® MYCOBACTERIUM KANSASII DNA) The specific detection of Mycobacterium abscessus DNA was confirmed using 100 or 20 copies of the control per reaction. Purified water was used as a negative sample to test for nonspecific amplification. The primer set used for Mycobacterium abscessus detection consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 21, a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 22, and a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 23. The probe used for Mycobacterium abscessus detection was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 20, synthesized according to standard methods (labeled at the 3' end with BODIPY-FL; see Figure 9 for the labeling position on the probe). Measurements were performed in duplicate, and the average fluorescence change rate was calculated.

[0141] (7-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 0.5 μM primer consisting of the base sequence shown in SEQ ID NO: 21 1.5 μM primer consisting of the base sequence shown in SEQ ID NO: 22 1.5 μM primer consisting of the base sequence shown in SEQ ID NO: 23 0.5 μM of the probe shown by SEQ ID NO: 20 (labeled at the 3' end with BODIPY-FL)

[0142] (7-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec) The measurement time in this example was approximately 30 minutes, enabling rapid detection of Mycobacterium abscessus DNA.

[0143] (7-4)Result Figure 4 shows a detection graph obtained when 100 copies of Mycobacterium abscessus DNA were detected by nucleic acid amplification and melting curve analysis. The measurement results for the primers and probes used in this test example are summarized in Table 19. The rate of change in fluorescence (represented as the quenching rate in this example) was calculated using the formula: (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0144] As shown in the results in Table 19, it was confirmed that the primers and probes used in the test example specifically detected only Mycobacterium abscessus DNA, and did not detect Mycobacterium tuberculosis DNA, Mycobacterium avium DNA, Mycobacterium intracellulare DNA, or Mycobacterium kansasii DNA. In addition, when purified water was used as a negative sample to check for non-specific amplification, no non-specific amplification was observed.

[0145] [Table 19]

[0146] Test Example 8: Detection of Mycobacterium kansasii (8-1) Method Mycobacterium tuberculosis DNA (Vircell, AMPLIRUN® MYCOBACTERIUM TUBERCULOSIS DNA CONTROL), Mycobacterium avium DNA (Vircell, AMPLIRUN® MYCOBACTERIUM AVIUM DNA CONTROL), Mycobacterium intracellulare DNA (Vircell, AMPLIRUN® MYCOBACTERIUM INTRACELLULARE DNA CONTROL), Mycobacterium abscessus DNA (ATCC, Genomic DNA from Mycobacterium abscessus strain L948 (ATCC19977)), Mycobacterium kansasii DNA (Vircell, AMPLIRUN® MYCOBACTERIUM KANSASII DNA) The specific detection of Mycobacterium kansasii DNA was confirmed by using 100 or 20 copies of the control per reaction. Purified water was used as a negative sample to check for nonspecific amplification. The primer set for detecting Mycobacterium kansasii consisted of a nucleic acid primer consisting of the base sequence shown in SEQ ID NO: 25 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 26. The probe for detecting Mycobacterium kansasii was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 24, synthesized according to standard methods (labeled at the 5' end with BODIPY-FL and phosphorylated at the 3' end; see Figure 10 for the labeling position on the probe). Measurements were performed in duplicate, and the average rate of fluorescence change was calculated.

[0147] (8-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 0.5 μM primer consisting of the base sequence shown in SEQ ID NO: 25 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 26 0.5 μM of the probe shown in SEQ ID NO: 24 (labeled with BODIPY-FL at the 5' end and phosphorylated at the 3' end)

[0148] (8-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec) The measurement time in this example was approximately 30 minutes, enabling rapid detection of Mycobacterium kansasii DNA.

[0149] (8-4)Result Figure 5 shows a detection graph obtained when 100 copies of Mycobacterium kansasii DNA were detected by nucleic acid amplification and melting curve analysis. The measurement results for the primers and probes used in this test example are summarized in Table 20. The rate of change in fluorescence (represented as the quenching rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0150] As shown in the results in Table 20, it was confirmed that the primers and probes used in the test example specifically detected only Mycobacterium kansasii DNA, and did not detect Mycobacterium tuberculosis DNA, Mycobacterium avium DNA, Mycobacterium intracellulare DNA, or Mycobacterium abscessus DNA. In addition, when purified water was used as a negative sample to check for non-specific amplification, no non-specific amplification was observed.

[0151] [Table 20]

[0152] Test Example 9: Comparison of detection sensitivity when targeting multi-copy or single-copy (9-1) Method To verify whether targeting a multicopy gene improves detection sensitivity compared to targeting a single-copy gene, 20, 10, and 5 copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA were used per reaction. Detection of Mycobacterium avium DNA and Mycobacterium intracellulare DNA was confirmed in reaction solution A containing primers and probes targeting multicopy genes and reaction solution B containing primers and probes targeting single-copy genes. The nucleic acid primer set used in reaction solution A (multicopy) was a set of nucleic acid primers consisting of the nucleotide sequence shown in SEQ ID NO: 15, 16, 18, and 19. The nucleic acid probes used were an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14, synthesized according to a standard method (labeled at the 3' end with BODIPY-FL; see Figure 7 for the labeling position in the probe), and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17, synthesized according to a standard method (labeled at the 5' end with CR6G and phosphorylated at the 3' end; see Figure 8 for the labeling position in the probe). The primers and probes for reaction solution B (single copy) were designed according to Non-Patent Document 5. The nucleic acid primer set used consisted of a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 28, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 29, a nucleic acid primer consisting of either the nucleotide sequence shown in SEQ ID NO: 31, and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 32. The nucleic acid probes used were an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27, synthesized according to a standard method (labeled at the 3' end with BODIPY-FL), and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 30, synthesized according to a standard method (labeled at the 5' end with CR6G and phosphorylated at the 3' end). Measurements were performed with N=4, and the detection rate was calculated.

[0153] (9-2) Reaction solution A (multi-copy) The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 16 0.5 μM of the probe shown in SEQ ID NO: 14 (labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 18 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 19 0.5 μM of the probe shown in SEQ ID NO: 17 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0154] (9-3) Reaction solution B (single copy copy) The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 28 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 29 0.5 μM of the probe shown by SEQ ID NO: 27 (labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 31 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 32 0.5 μM of the probe shown in SEQ ID NO: 30 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0155] (9-4) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (60 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0156] (9-5)Result Table 21 shows the fluorescence change rates observed when detecting Mycobacterium avium DNA and Mycobacterium intracellulare DNA in this example. Reaction Solution A (multicopy) demonstrated 100% detection of 5 to 20 copies of both Mycobacterium avium DNA and Mycobacterium intracellulare DNA. On the other hand, Reaction Solution B (single copy) demonstrated 100% detection of 20 copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA, but the detection rate decreased with decreasing concentration, reaching 75% at 10 copies and 50% at 5 copies. In other words, the detection sensitivity of Reaction Solution A (multicopy) was estimated to be more than four times that of Reaction Solution B (single copy). The results of this example confirmed that using a multicopy region based on the present invention as the target gene enables more sensitive detection than using a single-copy region as the target gene.

[0157] [Table 21]

[0158] Test Example 10: Confirmatory test for simultaneous detection of Mycobacterium avium and Mycobacterium intracellulare (10-1) Method Twenty copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA were used per reaction to confirm the simultaneous detection of Mycobacterium avium DNA and Mycobacterium intracellulare DNA. Purified water was used as a negative sample to test for nonspecific amplification. The primer set for Mycobacterium avium detection consisted of a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 15 and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 44. The primer set for Mycobacterium intracellulare detection consisted of a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 57 and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 64. The Mycobacterium avium detection probe was an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 33, synthesized according to standard methods (labeled at the 3' end with BODIPY-FL; see Figure 13 for the labeling position on the probe). The probe used for detecting Mycobacterium intracellulare was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37, synthesized according to a standard method (the 5' end was labeled with CR6G and the 3' end was phosphorylated. See Figure 14 for the labeling position in the probe). Measurements were performed with N=4, and the average rate of fluorescence change was calculated.

[0159] (10-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 44 0.5 μM of the probe shown in SEQ ID NO: 33 (labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 57 0.3 μM Primer consisting of the base sequence shown in SEQ ID NO: 64 0.45 μM of the probe shown in SEQ ID NO: 37 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0160] (10-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec) The measurement time in this example was approximately 30 minutes, and rapid detection of Mycobacterium avium DNA and Mycobacterium intracellulare DNA was possible.

[0161] (10-4)Result Figure 11 shows a detection graph obtained when 20 copies of Mycobacterium avium DNA were detected by nucleic acid amplification and melting curve analysis. Figure 12 shows a detection graph obtained when 20 copies of Mycobacterium intracellulare DNA were detected. Similarly, the measurement results using the probe used in this test example are summarized in Table 22. The fluorescence change rate (represented as the quenching rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0162] As shown in the results in Table 22, it was demonstrated that simultaneous detection of Mycobacterium avium DNA and Mycobacterium intracellulare DNA is possible. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed with either probe.

[0163] [Table 22]

[0164] Test Example 11: Examination of probes for detecting Mycobacterium avium (11-1) Method To search for a probe capable of specifically detecting Mycobacterium avium, 20 copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA were used per reaction, and five nucleic acid probes designed based on the 30th to 70th bases of SEQ ID NO: 4 were used to confirm the presence or absence of nonspecific amplification and the detection of Mycobacterium avium DNA. Purified water was used as a negative sample for the nonspecific amplification assay. The nucleic acid primer set used consisted of a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 15, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 44, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 57, and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 64. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 14, 33, 34, 35, and 36, synthesized according to standard methods (all labeled at the 3' end with BODIPY-FL; see Figure 13 for the labeling position in each probe), and oligonucleotides consisting of the base sequence shown in SEQ ID NO: 37, synthesized according to standard methods (labeled at the 5' end with CR6G and phosphorylated at the 3' end; see Figure 14 for the labeling position in the probe). Measurements were performed with N=4, and the average values ​​of the detection temperature and fluorescence change rate were calculated.

[0165] (11-2) Reaction solution The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 44 0.5 μM Probe consisting of any of the base sequences shown in SEQ ID NOs: 14, 33, 34, 35, and 36 (all labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 57 0.3 μM Primer consisting of the base sequence shown in SEQ ID NO: 64 0.45 μM Probe consisting of the base sequence shown in SEQ ID NO: 37 (5' end labeled with CR6G and 3' end phosphorylated)

[0166] (11-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0167] (11-4)Result Table 23 shows the probes confirmed in this example to specifically detect Mycobacterium avium, as well as the detection temperatures and fluorescence change rates when detecting Mycobacterium avium DNA and Mycobacterium intracellulare DNA. As shown in these results, all probes specifically detected Mycobacterium avium DNA.

[0168] [Table 23]

[0169] Test Example 12: Examination of probes for detecting Mycobacterium intracellulare (12-1) Method To search for a probe capable of specifically detecting Mycobacterium intracellulare, 20 copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA were used per reaction. Six nucleic acid probes designed based on the 45th to 85th bases of SEQ ID NO: 6 were used to confirm the presence or absence of nonspecific amplification and the detection of Mycobacterium avium DNA. Purified water was used as a negative sample for the nonspecific amplification assay. The nucleic acid primer set used consisted of a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 15, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 44, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 57, and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 64. The nucleic acid probes used were an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, synthesized according to a standard method (labeled at the 3' end with BODIPY-FL; see Figure 13 for the labeling position in the probe), and an oligonucleotide consisting of the base sequence shown in any of SEQ ID NOs: 17, 230607JP017, 38, 39, 40, 41, and 42, synthesized according to a standard method (labeled at only either the 5' or 3' end with CR6G; when the 5' end was labeled with CR6G, the 3' end was phosphorylated; see Figure 14 for the labeling position in each probe). Measurements were performed with N=4, and the average detection temperature and fluorescence change rate were calculated.

[0170] (12-2) Reaction solution The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 44 0.5 μM probe shown in SEQ ID NO: 14 (labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 57 0.3 μM Primer consisting of the base sequence shown in SEQ ID NO: 64 0.45 μM probe consisting of any of the base sequences shown in SEQ ID NOs: 17, 37, 38, 39, 40, 41, and 42 (SEQ ID NOs: 17, 37, 38, 39, and 40 are labeled with CR6G at the 5' end and phosphorylated at the 3' end; SEQ ID NOs: 41 and 42 are labeled with CR6G at the 3' end)

[0171] (12-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0172] (12-4)Result Table 24 shows the probes confirmed in this example to specifically detect Mycobacterium intracellulare, as well as the detection temperatures and fluorescence change rates when detecting Mycobacterium avium DNA and Mycobacterium intracellulare DNA. As shown in these results, all probes specifically detected Mycobacterium intracellulare DNA.

[0173] [Table 24]

[0174] Test Example 13: Examination of primers for detecting Mycobacterium avium (13-1) Method To search for primers capable of specifically detecting Mycobacterium avium, 20 copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA were used per reaction, and a total of 12 nucleic acid primers designed based on the nucleotide sequence shown in SEQ ID NO: 4 were used to confirm the presence or absence of nonspecific amplification and the detection of Mycobacterium avium DNA. Purified water was used as a negative sample for the nonspecific amplification assay. The nucleic acid primer sets used included a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 15 or 43, a nucleic acid primer consisting of any of the nucleotide sequences shown in SEQ ID NOs: 16 and 44-54, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 57, and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 64. The nucleic acid probes used were an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, synthesized according to a standard method (labeled at the 3' end with BODIPY-FL; see Figure 13 for the labeling position in the probe), and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, synthesized according to a standard method (labeled at the 5' end with CR6G and phosphorylated at the 3' end; see Figure 14 for the labeling position in the probe). Measurements were performed with N=4, and the average rate of fluorescence change was calculated.

[0175] (13-2) Reaction solution The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM Primer consisting of the base sequence shown in SEQ ID NO: 15 or 43 3.0 μM Primer consisting of the base sequence shown in any one of SEQ ID NOs: 16 and 44 to 54 0.5 μM probe shown in SEQ ID NO: 14 (labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 57 0.3 μM Primer consisting of the base sequence shown in SEQ ID NO: 64 0.45 μM of the probe shown in SEQ ID NO: 17 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0176] (13-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0177] (13-4)Result Table 25 shows the primers confirmed in this example to specifically detect Mycobacterium avium, as well as the rate of fluorescence change when Mycobacterium avium DNA and Mycobacterium intracellulare DNA were detected. As shown in the results, Mycobacterium avium DNA was specifically detected with all primers.

[0178] [Table 25]

[0179] Test Example 14: Examination of primers for detecting Mycobacterium intracellulare (14-1) Method To search for primers capable of specifically detecting Mycobacterium intracellulare, 20 copies of Mycobacterium avium DNA and Mycobacterium intracellulare DNA were used per reaction. A total of 11 nucleic acid primer combinations designed based on the nucleotide sequence shown in SEQ ID NO: 6 were used to confirm the presence or absence of nonspecific amplification and the detection of Mycobacterium intracellulare DNA. Purified water was used as a negative sample for the nonspecific amplification assay. The nucleic acid primer sets used included a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 15, a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 16, a nucleic acid primer consisting of the nucleotide sequences shown in SEQ ID NO: 18 and any of SEQ ID NOs: 55 to 63, and a nucleic acid primer consisting of the nucleotide sequence shown in SEQ ID NO: 19 or 64. The nucleic acid probes used were an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, synthesized according to a standard method (labeled at the 3' end with BODIPY-FL; see Figure 13 for the labeling position in the probe), and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, synthesized according to a standard method (labeled at the 5' end with CR6G and phosphorylated at the 3' end; see Figure 14 for the labeling position in the probe). Measurements were performed with N=4, and the average rate of fluorescence change was calculated.

[0180] (14-2) Reaction solution The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM primer consisting of the base sequence shown in SEQ ID NO: 16 0.5 μM of the probe shown in SEQ ID NO: 14 (labeled at the 3' end with BODIPY-FL) 3.0 μM of a primer consisting of a base sequence shown in any one of SEQ ID NOs: 18 and 55 to 63 0.3 μM Primer consisting of the base sequence shown in SEQ ID NO: 19 or 64 0.45 μM of the probe shown in SEQ ID NO: 17 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0181] (14-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0182] (14-4)Result Table 26 shows the primers confirmed in this example to specifically detect Mycobacterium intracellulare, as well as the rate of fluorescence change when Mycobacterium avium DNA and Mycobacterium intracellulare DNA were detected. As shown in these results, all primers specifically detected Mycobacterium intracellulare DNA.

[0183] [Table 26]

[0184] Test Example 15: Confirmatory test for simultaneous detection of Mycobacterium avium and Mycobacterium intracellulare using NALC-treated sputum specimens (15-1) Method Based on the results obtained so far, we verified the simultaneous detection of Mycobacterium avium and Mycobacterium intracellulare. Ten NALC-treated sputum samples were pooled to prepare pooled samples. Then, 300 μL of NALC-treated sputum sample was added to Easy-Beads (Toyobo) containing 1 mL of sterile purified water and centrifuged at 13,000 rpm for 3 minutes. The supernatant was then removed to leave a residual liquid of 100 μL, and 50 μL of lysis solution was added. After heating at 95°C for 10 minutes, the samples were disrupted at 2850 rpm for 3 minutes using a DISRUPTOR GENIE (MS Equipment). After further centrifugation at 13,000 rpm for 3 minutes, 50 μL of the supernatant was used for measurement. In this study, 50 μL of the supernatant was mixed with 250 copies each of Mycobacterium avium DNA and Mycobacterium intracellulare DNA (20 copies per reaction). Purified water was used as a negative sample for the measurement to check for nonspecific amplification. The nucleic acid primer sets are listed in Table 27. As nucleic acid probes, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, synthesized according to a standard method (labeled with BODIPY-FL at the 3' end; see Figure 13 for the labeling position in the probe), and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, synthesized according to a standard method (labeled with CR6G at the 5' end and phosphorylated at the 3' end; see Figure 14 for the labeling position in the probe). Measurements were performed in four sets, and the average detection temperature and fluorescence change rate were calculated.

[0185] (15-2) Reaction solution The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The amounts of GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) were adjusted according to the instructions in each instruction manual. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 15 3.0 μM Primer consisting of the base sequence shown in any one of SEQ ID NOs: 16, 44, and 45 0.5 μM probe shown in SEQ ID NO: 14 (labeled at the 3' end with BODIPY-FL) 3.0 μM primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 18, 55, and 56 0.3 μM primer consisting of the base sequence shown in SEQ ID NO: 19 0.45 μM of the probe shown in SEQ ID NO: 17 (labeled with CR6G at the 5' end and phosphorylated at the 3' end)

[0186] (15-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity was measured in each cycle. (Nucleic acid amplification and melting curve analysis) 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0187] (15-4)Result Table 27 shows the fluorescence change rates observed when detecting Mycobacterium avium DNA and Mycobacterium intracellulare DNA in this example. As shown in these results, all primer combinations specifically detected Mycobacterium avium and Mycobacterium intracellulare DNA in the NALC-treated pooled sputum samples. Furthermore, when nonspecific amplification was confirmed using purified water as a negative sample, no nonspecific amplification was observed with any of the probes. The NALC-treated sputum samples used in this example did not undergo a nucleic acid purification process and therefore contained biological contaminants, such as various proteins, that could inhibit nucleic acid amplification reactions. The results of this example confirmed that the present invention can be used to perform high-sensitivity assays without any problems on samples treated with NALC-treated sputum samples.

[0188] [Table 27] [Industrial Applicability]

[0189] The use of the primers and probes of the present invention enables specific, highly sensitive, and complete detection of Mycobacterium species, which is believed to enable rapid and reliable differentiation of Mycobacterium species, leading to early diagnosis and treatment of nontuberculous mycobacterial disease.

Claims

1. 1. A method for detecting Mycobacterium species that may be contained in a sample, the method comprising amplifying multiple nucleic acid regions derived from a multicopy gene of Mycobacterium species, wherein the multicopy gene has the following characteristics (a) and / or (b): (a) the sequence identity between strains of the Mycobacterium species to be detected is 82% or more, and the sequence identity between the Mycobacterium species to be detected and other Mycobacterium species is less than 82%; (b) present in more than 98% of strains of Mycobacterium species detected;

2. 2. The method of claim 1, wherein the multicopy gene has characteristics (a) and (b).

3. The method of claim 1, wherein each of the plurality of nucleic acid regions independently has a length of 50 to 1500 bases.

4. The method of claim 1, wherein the sequence identity of the plurality of nucleic acid regions is 85% or more.

5. The method of claim 1, wherein the multicopy gene is an HNH nuclease gene.

6. The method of claim 1, wherein each of the plurality of nucleic acid regions exhibits 85% or more sequence identity to any of the base sequence shown in SEQ ID NO: 1 or its complementary base sequence, the base sequence shown in SEQ ID NO: 3 or its complementary base sequence, the base sequence shown in SEQ ID NO: 5 or its complementary base sequence, the base sequence shown in SEQ ID NO: 7 or its complementary base sequence, and the base sequence shown in SEQ ID NO: 9 or its complementary base sequence.

7. The method of claim 1, wherein each of the multiple nucleic acid regions exhibits 85% or more sequence identity to any of the base sequence shown in SEQ ID NO: 2 or its complementary base sequence, SEQ ID NO: 4 or its complementary base sequence, SEQ ID NO: 6 or its complementary base sequence, SEQ ID NO: 8 or its complementary base sequence, and SEQ ID NO: 10 or its complementary base sequence.

8. 2. The method of claim 1, wherein the Mycobacterium species is one or more of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium kansasii.

9. A probe for detecting Mycobacterium genus acid-fast bacteria, having any one of the following characteristics (A) to (E) and the following characteristic (F): (A) a base sequence A1 of at least 10 consecutive bases in the base sequence of 50 to 90 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence A2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence A1; (B) a base sequence B1 of at least 10 consecutive bases in the base sequence of positions 30 to 70 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence B2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence B1; (C) a base sequence C1 of at least 10 consecutive bases in the base sequence of positions 45 to 85 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence C2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence C1; (D) a base sequence D1 of at least 10 consecutive bases in the base sequence of positions 45 to 85 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence D2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence D1; (E) a base sequence E1 of at least 10 consecutive bases in the base sequence of positions 45 to 85 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence E2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence E1; (F) Only either the 5' or 3' end is labeled.

10. 10. The probe according to claim 9, wherein the length of the base sequence of (A) to (E) is 12 to 20 bases.

11. The probe according to claim 9, wherein the base sequence of (A) comprises the base sequence shown in SEQ ID NO: 11 or a complementary base sequence thereof, the base sequence of (B) comprises the base sequence shown in any one of SEQ ID NOs: 14, 33, 34, 35, and 36 or a complementary base sequence thereof, the base sequence of (C) comprises the base sequence shown in any one of SEQ ID NOs: 17, 37, 38, 39, 40, 41, and 42 or a complementary base sequence thereof, the base sequence of (D) comprises the base sequence shown in SEQ ID NO: 20 or a complementary base sequence thereof, and the base sequence of (E) comprises the base sequence shown in SEQ ID NO: 24 or a complementary base sequence thereof.

12. The probe according to claim 9, wherein the label (F) is a label with a fluorescence quenching dye that is quenched by interaction with guanine when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe, and the labeled terminal base is cytosine.

13. The probe according to claim 9, wherein the label (F) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

14. 10. A method for detecting Mycobacterium species that may be contained in a sample, using one or more probes according to claim 9.

15. The following steps (1), (2), and (3): (1) providing a sample that may contain Mycobacterium species; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained by the nucleic acid amplification reaction of step (2) using the one or more probes; 15. The method of claim 14, comprising:

16. 16. The method of claim 15, wherein the Mycobacterium species is one or more of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium kansasii.

17. 16. The method according to claim 15, wherein the step (2) is carried out by PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.

18. The method according to claim 17, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a mutant thereof.

19. The method according to claim 15, wherein the step (2) is carried out using a primer set having any one of the following characteristics (G) to (K): (G) A primer comprising: a first primer having a base sequence S1 of at least 20 consecutive bases in the base sequence of positions 1 to 65 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1; and a second primer having a base sequence S3 of at least 20 consecutive bases in the base sequence of positions 65 to 130 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second primer is complementary to a DNA extension product of the first primer; (H) A first primer having a base sequence S5 of at least 20 consecutive bases in the base sequence of positions 1 to 60 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S5, and a second primer having a base sequence S7 of at least 20 consecutive bases in the base sequence of positions 60 to 120 of SEQ ID NO: 4 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second primer is complementary to a DNA extension product of the first primer; (I) A first primer having a base sequence S9 of at least 20 consecutive bases in the base sequence of positions 1 to 60 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence S10 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S9, and a second primer having a base sequence S11 of at least 20 consecutive bases in the base sequence of positions 60 to 120 of SEQ ID NO: 6 or a complementary base sequence thereof, or a base sequence S12 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S11, wherein the second primer is complementary to a DNA extension product of the first primer; (J) a first primer having a base sequence S13 of at least 20 consecutive bases in the base sequence of positions 1 to 60 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S14 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S13; and a second primer having a base sequence S15 of at least 20 consecutive bases in the base sequence of positions 60 to 114 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S16 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S15, wherein the second primer is complementary to a DNA extension product of the first primer; (K) A first primer having a base sequence S17 of at least 20 consecutive bases in the base sequence from positions 1 to 65 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence S18 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S17; and a second primer having a base sequence S19 of at least 20 consecutive bases in the base sequence from positions 65 to 130 of SEQ ID NO: 10 or a complementary base sequence thereof, or a base sequence S20 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S19, wherein the second primer is complementary to a DNA extension product of the first primer.

20. the primer set having the characteristic (G) comprises the first primer having the nucleotide sequence shown in SEQ ID NO: 12, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substituting, deleting, inserting or adding 1 to 3 bases in said nucleotide sequence, and the second primer having the nucleotide sequence shown in SEQ ID NO: 13, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substituting, deleting, inserting or adding 1 to 3 bases in said nucleotide sequence; the primer set having the characteristic (H) comprises the first primer having a nucleotide sequence shown in SEQ ID NO: 15 or 43, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added, and the second primer having a nucleotide sequence shown in any of SEQ ID NOs: 16, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, a complementary nucleotide sequence thereof, or a nucleotide sequence of said nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added; the primer set having the characteristic (I) comprises the first primer having a nucleotide sequence shown in any one of SEQ ID NOs: 18, 55, 56, 57, 58, 59, 60, 61, 62, and 63, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substitution, deletion, insertion, or addition of 1 to 3 bases in said nucleotide sequence, and the second primer having a nucleotide sequence shown in SEQ ID NO: 19 or 64, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substitution, deletion, insertion, or addition of 1 to 3 bases in said nucleotide sequence; the primer set having the characteristic (J) comprises the first primer having the nucleotide sequence shown in SEQ ID NO: 21, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substituting, deleting, inserting or adding 1 to 3 bases in said nucleotide sequence, and the second primer having the nucleotide sequence shown in SEQ ID NO: 22 or 23, a complementary nucleotide sequence thereof, or a nucleotide sequence obtained by substituting, deleting, inserting or adding 1 to 3 bases in said nucleotide sequence; The method of claim 19, wherein the primer set having characteristic (K) comprises the first primer having the nucleotide sequence shown in SEQ ID NO: 25, a complementary nucleotide sequence thereof, or a nucleotide sequence of either of these nucleotide sequences in which 1 to 3 bases have been substituted, deleted, inserted or added; and the second primer having the nucleotide sequence shown in SEQ ID NO: 26, a complementary nucleotide sequence thereof, or a nucleotide sequence of either of these nucleotide sequences in which 1 to 3 bases have been substituted, deleted, inserted or added.

21. 16. The method of claim 15, wherein the detecting step in step (3) is performed by melting curve analysis.

22. A reagent or kit for detecting Mycobacterium species, for use in the method according to any one of claims 1 to 8 and 14 to 21.

23. A reagent or kit for detecting Mycobacterium species, comprising at least one probe according to any one of claims 9 to 13 and / or at least one primer set according to claim 19 or 20.

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