Oligonucleotide for detecting mycobacterium tuberculosis complex, and application of the same

Abstract

To provide a probe that can specifically detect mycobacterium tuberculosis complex.SOLUTION: A probe for detecting mycobacterium tuberculosis complex has an oligonucleotide including (A) 79th to 130th or (B) 277th to 312th base sequence of a specific base sequence, or (C) 37th to 84th base sequence of a specific base sequence or a base sequence of 15 bases or more and 24 bases or less continuous in a base sequence complementary with them, or a base sequence having 1 to 3 bases of difference from the base sequence, where a terminal base of at least one of 5' end and 3' end is cytosine, cytosine of a terminal base of one of 5' end and 3' end has fluorescent dye labeled with oligonucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a probe for specifically detecting Mycobacterium tuberculosis complex, and a detection method, reagent, kit, etc. that uses the probe. [Background technology]

[0002] Tuberculosis is an infectious disease caused by the Mycobacterium tuberculosis (MTB) and other bacteria of the MTB complex. People who develop tuberculosis primarily experience respiratory symptoms. Furthermore, when tuberculosis patients expel MTB complex bacteria into the air by coughing or sneezing, the MTB complex can spread to others through the air. Because MTB complex bacteria are highly contagious, rapid and accurate testing of people infected with MTB is extremely important for public health.

[0003] Culture identification has long been used as a testing method for the Mycobacterium tuberculosis complex, but recently nucleic acid amplification has also been used. The main nucleic acid amplification methods used include PCR, real-time PCR, and TRC (Patent Document 1, Non-Patent Document 1, Patent Document 2). Examples of nucleic acids targeted by nucleic acid amplification include ribosomal RNA genes and rpoB genes. Furthermore, the IS6110 region, a multicopy region specific to the Mycobacterium tuberculosis complex, is one of the targets that has been widely used in in-house testing in laboratories and medical facilities (Non-Patent Document 2).

[0004] It is said that there are approximately 20 copies of IS6110 in the genomic DNA of M. tuberculosis complex. However, some M. tuberculosis complex strains lack IS6110. Furthermore, the sequence of the IS6110 region contains single nucleotide polymorphisms, meaning that there are positions where the bases may differ between strains. Therefore, conventional methods for detecting M. tuberculosis complex that target IS6110 may not be able to detect IS6110-deficient strains or strains with single nucleotide polymorphisms in this region.

[0005] Conventional detection methods targeting the IS6110 region mainly involve real-time PCR using double-labeled nucleic acid probes (also called hydrolysis probes) such as TaqMan probes or molecular beacon probes (Non-Patent Documents 1 and 2). This method allows for the quantification of viral load based on control RNA, but requires photometry for each PCR cycle, often requiring approximately one hour for detection even at the shortest possible time.

[0006] Another method for detecting nucleic acid amplification products is melting curve analysis. Melting curve analysis allows nucleic acid amplification and detection to be performed in separate steps, making measurements relatively simple and requiring as little as 30 minutes. Furthermore, melting curve analysis using fluorescently labeled nucleic acid probes has the advantage of being easily adaptable to genetic testing using automated analyzers. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4905130 [Patent Document 2] Patent No. 6142524 [Non-patent literature]

[0008] [Non-Patent Document 1] BMMelissa et al., Journal of Clinical Microbiology, 2011, 49, p.3458-3462 [Non-patent document 2] HY.Wang et al., Scientific Reports, 2019, 14;9(1).113 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a further useful method for specifically detecting the tubercle bacillus complex. [Means for solving the problem]

[0010] In view of the above problems, the present inventors conducted extensive research and found that Mycobacterium tuberculosis complex can be specifically detected by using a probe in which an oligonucleotide consisting of a specific base sequence is fluorescently labeled by a predetermined method. Further research led to the discovery that Mycobacterium tuberculosis complex can be accurately detected with even higher specificity by using two or more of these probes in combination. Based on the above findings, the present inventors conducted further research and completed the present invention.

[0011] Representative aspects of the present invention include the following. [Section 1] A probe for detecting Mycobacterium tuberculosis complex, comprising any one of the following oligonucleotides (A) to (C): (A) an oligonucleotide comprising a base sequence of 15 to 24 consecutive bases in the base sequence of positions 79 to 130 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is a cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye; (B) an oligonucleotide comprising a base sequence of 15 to 24 consecutive bases in the base sequence from positions 277 to 312 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence differing from said base sequence by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye; (C) An oligonucleotide comprising a base sequence of 15 to 24 consecutive bases in the base sequence 37 to 84 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence differing from said base sequence by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye. [Section 2] Item 2. The probe for detecting a Mycobacterium tuberculosis complex according to Item 1, wherein the oligonucleotides (A) to (C) are 19 to 24 bases in length. [Section 3] Item 2. The probe for detecting a Mycobacterium tuberculosis complex according to Item 1, wherein the oligonucleotides (A) to (C) are 15 to 19 bases in length. [Section 4] the oligonucleotide (A) comprises a nucleotide sequence shown in any one of SEQ ID NOs: 23 to 28 or a nucleotide sequence complementary thereto; the oligonucleotide (B) comprises a nucleotide sequence shown in any one of SEQ ID NOs: 29 to 31 or a nucleotide sequence complementary thereto; Item 4. The probe for detecting a Mycobacterium tuberculosis complex according to any one of Items 1 to 3, wherein the oligonucleotide (C) comprises a base sequence shown in any one of SEQ ID NOs: 48 to 56. [Section 5] Item 5. The probe for detecting a Mycobacterium tuberculosis complex according to any one of Items 1 to 4, wherein the fluorescent dye is a fluorescence quenching dye that is quenched by interaction with guanine. [Section 6] Item 6. The probe for detecting Mycobacterium tuberculosis complex according to any one of Items 1 to 5, wherein the fluorescent dye is 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. [Section 7] Item 7. The probe for detecting Mycobacterium tuberculosis complex according to any one of Items 1 to 6, wherein the fluorescent dye is 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 8] A probe set for detecting the tuberculosis complex, comprising at least two probes selected from the probes for detecting the tuberculosis complex, each having the oligonucleotide (A) to (C) according to any one of Items 1 to 7. [Section 9] Item 9. A probe set for detecting mycobacterium tuberculosis complex according to Item 8, comprising at least one probe selected from the group consisting of a probe for detecting mycobacterium tuberculosis complex having the oligonucleotide (A) and a probe for detecting mycobacterium tuberculosis complex having the oligonucleotide (B), and at least one probe selected from the group consisting of a probe for detecting mycobacterium tuberculosis complex having the oligonucleotide (C). [Section 10] Item 10. A probe set for detecting mycobacterium tuberculosis complex according to Item 8 or 9, comprising at least one probe selected from the probes for detecting mycobacterium tuberculosis complex having the oligonucleotide (B) and at least one probe selected from the probes for detecting mycobacterium tuberculosis complex having the oligonucleotide (C). [Section 11] A primer for amplifying nucleic acid of Mycobacterium tuberculosis complex, which is used in combination with the probe for detecting Mycobacterium tuberculosis complex according to any one of Items 1 to 7 or the probe set for detecting Mycobacterium tuberculosis complex according to any one of Items 8 to 10, and which comprises any one of the following oligonucleotides (E) to (J): (E) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the 50th to 90th nucleotides of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (F) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 115 to 205 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (G) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 230 to 280 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (H) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence of positions 355 to 400 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence that differs from said base sequence by 1 to 3 bases. [Section 12] the oligonucleotide (E) comprises a base sequence shown in any one of SEQ ID NOs: 1 to 3, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; the oligonucleotide (F) comprises a base sequence shown in any one of SEQ ID NOs: 13 to 16, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; the oligonucleotide (G) comprises a base sequence shown in any one of SEQ ID NOs: 4 to 12, a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases; the oligonucleotide (H) comprises a base sequence shown in any one of SEQ ID NOs: 17 to 22, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; the oligonucleotide (I) comprises a base sequence shown in any one of SEQ ID NOs: 33 to 37, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; Item 12. The primer for amplifying Mycobacterium tuberculosis complex nucleic acid according to Item 11, wherein the oligonucleotide (J) comprises a nucleotide sequence shown in any one of SEQ ID NOs: 38 to 47, a nucleotide sequence complementary thereto, or a nucleotide sequence differing from the nucleotide sequence by 1 to 3 nucleotides. [Section 13] Item 13. The primer for amplifying Mycobacterium tuberculosis complex nucleic acid according to Item 11 or 12, which is a PCR primer. [Section 14] A primer set for amplifying Mycobacterium tuberculosis complex nucleic acid, which is used in combination with the probe for detecting Mycobacterium tuberculosis complex according to any one of Items 1 to 7 or the probe set for detecting Mycobacterium tuberculosis complex according to any one of Items 8 to 10, and which comprises at least one combination selected from the group consisting of a combination of a primer having the following oligonucleotide (E) and a primer having the following oligonucleotide (F); a combination of a primer having the following oligonucleotide (G) and a primer having the following oligonucleotide (H); and a combination of a primer having the following oligonucleotide (I) and a primer having the following oligonucleotide (J): (E) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the 50th to 90th nucleotides of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (F) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 115 to 205 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (G) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 230 to 280 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (H) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence of positions 355 to 400 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence that differs from said base sequence by 1 to 3 bases. [Section 15] The following steps (1), (2), and (3): (1) providing a sample that may contain the Mycobacterium tuberculosis complex; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting the one or more nucleic acid amplification products of step (2) using one or more probes or probe sets; A method for detecting a tuberculosis complex, comprising: the step of: detecting a tuberculosis complex; wherein the probe or probe set is the probe for detecting a tuberculosis complex according to any one of Items 1 to 7; or the probe set for detecting a tuberculosis complex according to any one of Items 8 to 10. [Section 16] Item 16. The detection method according to Item 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. [Section 17] Item 17. The detection method according to Item 16, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Section 18] Item 18. The method for detecting Mycobacterium tuberculosis complex nucleic acid according to any one of Items 15 to 17, wherein the nucleic acid amplification reaction in step (2) uses a primer set for amplifying Mycobacterium tuberculosis complex nucleic acid, the primer set comprising at least one combination selected from the group consisting of a combination of a primer having the following oligonucleotide (E) and a primer having the following oligonucleotide (F); a combination of a primer having the following oligonucleotide (G) and a primer having the following oligonucleotide (H); and a combination of a primer having the following oligonucleotide (I) and a primer having the following oligonucleotide (J): (E) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the 50th to 90th nucleotides of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (F) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 115 to 205 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (G) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 230 to 280 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (H) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence of positions 355 to 400 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence that differs from said base sequence by 1 to 3 bases. [Section 19] A reagent for detecting the tuberculosis complex, comprising the probe for detecting the tuberculosis complex according to any one of Items 1 to 7 or the probe set for detecting the tuberculosis complex according to any one of Items 8 to 10. [Section 20] Item 20. A kit for detecting mycobacterium tuberculosis complex, comprising the reagent for detecting mycobacterium tuberculosis complex according to Item 19. [Section 21] Item 21. The kit for detecting Mycobacterium tuberculosis complex according to Item 20, further comprising a primer for amplifying nucleic acid of Mycobacterium tuberculosis complex and a DNA polymerase. [Section 22] Item 22. The kit for detecting Mycobacterium tuberculosis complex according to Item 21, wherein the primers for amplifying Mycobacterium tuberculosis complex nucleic acids comprise at least one combination selected from the group consisting of a combination of a primer having the following oligonucleotide (E) and a primer having the following oligonucleotide (F); a combination of a primer having the following oligonucleotide (G) and a primer having the following oligonucleotide (H); and a combination of a primer having the following oligonucleotide (I) and a primer having the following oligonucleotide (J): (E) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the 50th to 90th nucleotides of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (F) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 115 to 205 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (G) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence from positions 230 to 280 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (H) an oligonucleotide comprising a nucleotide sequence of 23 to 28 consecutive nucleotides in the nucleotide sequence of positions 355 to 400 of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, or comprising a nucleotide sequence which differs from said nucleotide sequence by 1 to 3 nucleotides; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence that differs from said base sequence by 1 to 3 bases. [Effects of the Invention]

[0012] The present invention can provide a further useful method for specifically detecting Mycobacterium tuberculosis complex, in particular a method for specifically detecting Mycobacterium tuberculosis complex in a short period of time with high sensitivity while targeting the IS6110 region, which is a multicopy region, and further a method that can detect IS6110-deficient strains and strains with single nucleotide polymorphisms in the region, thereby making a significant contribution to the field of clinical diagnosis. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows the results of melting curve analysis carried out in Example 1. 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] In one embodiment, the present invention provides a method for detecting the Mycobacterium tuberculosis complex using a labeled probe having a specific base sequence. This method is capable of specifically detecting the Mycobacterium tuberculosis complex and is also capable of detecting it with high sensitivity in a short period of time. The M. tuberculosis complex is known as a collective term for M. tuberculosis and its related bacteria (including M. bovis, M. africanum, and M. microti). The labeled probe is designed to target a specific region in the genomic DNA of the Mycobacterium tuberculosis complex, thereby enabling reliable detection of the Mycobacterium tuberculosis complex, for example, by melting curve analysis. In this specification, SEQ ID NOs: 57 and 58 are base sequences corresponding to the genomic DNA sequence of the Mycobacterium tuberculosis complex. SEQ ID NO: 57 represents a partial sequence of the IS6110 region, and SEQ ID NO: 58 represents a partial sequence of the dnaJ gene of M. tuberculosis. The labeled probe is preferably a fluorescently labeled probe having a specific base sequence that targets a specific region of the base sequence shown in SEQ ID NO: 57 or 58. Furthermore, by using a probe set that combines two or more types of the fluorescently labeled probe, it becomes possible to detect the Mycobacterium tuberculosis complex with higher sensitivity and accuracy.

[0017] In one aspect, the probe for detecting Mycobacterium tuberculosis complex of the present invention comprises any one of the following oligonucleotides (A) to (C): (A) an oligonucleotide comprising a base sequence A1 of 15 to 24 consecutive bases in the base sequence from positions 79 to 130 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence A2 which differs from the base sequence A1 by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye; (B) an oligonucleotide comprising a base sequence B1 of 15 to 24 consecutive bases in the base sequence from positions 277 to 312 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence B2 which differs from said base sequence B1 by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye; (C) An oligonucleotide comprising a base sequence C1 of 15 to 24 consecutive bases in the base sequence 37 to 84 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence C2 which differs from the base sequence C1 by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye. The probe for detecting Mycobacterium tuberculosis complex of the present invention may be used singly or, preferably, in combination of two or more types.

[0018] One embodiment of the present invention is a method for detecting Mycobacterium tuberculosis complex that may be contained in a sample. The detection method uses the above-mentioned probe for detecting Mycobacterium tuberculosis complex. In a specific embodiment, by using one probe for detecting Mycobacterium tuberculosis complex or a combination (probe set) of two or more probes for detecting Mycobacterium tuberculosis complex in a reaction solution, Mycobacterium tuberculosis complex 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).

[0019] In a particular embodiment, the method for detecting the Mycobacterium tuberculosis complex comprises at least the following steps (i) and (ii): (i) carrying out a nucleic acid amplification reaction using one or more primer sets for nucleic acid amplification of the Mycobacterium tuberculosis complex having specific base sequences and a specific region present in the Mycobacterium tuberculosis complex as a template to generate one or more nucleic acid amplification products; and (ii) detecting one or more nucleic acid amplification products obtained in step (i) using one or more of the probes or probe sets for detecting Mycobacterium tuberculosis complex; Preferably, the method comprises the steps of: (i) carrying out a PCR reaction; and (ii) carrying out a melting curve analysis. More preferably, the method is a PCR-melting curve analysis method in which a PCR reaction is carried out in step (i) and a melting curve analysis is carried out in step (ii). Steps (i) and (ii) may be carried out in the same reaction solution. Steps (i) and (ii) may also be carried out consecutively or simultaneously.

[0020] In a particular embodiment, the method for detecting Mycobacterium tuberculosis complex comprises the following steps (1), (2), and (3): (1) providing a sample that may contain the Mycobacterium tuberculosis complex; (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 of step (2) using one or more of the probes or probe sets for detecting Mycobacterium tuberculosis complex; Preferably, the detection method comprises carrying out step (2) by PCR reaction and step (3) by melting curve analysis (PCR-melting curve analysis method). Steps (2) and (3) may be carried out in the same reaction solution. Steps (2) and (3) may also be carried out consecutively or simultaneously.

[0021] [Process (1)] In one embodiment, step (1) is a step of preparing a sample that may contain the Mycobacterium tuberculosis complex (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 the Mycobacterium tuberculosis complex. Examples include, but are not limited to, sputum, blood, urine, pus, cerebrospinal fluid, pleural effusion, ascites, gastric fluid, pharyngeal swabs, nasal swabs, saliva, oral scrapings, bronchial lavage fluid, alveolar lavage fluid, tissue sections, skin, vomit, and feces collected from a subject suspected of infection with the Mycobacterium tuberculosis complex, as well as isolated culture colonies and liquid culture fluids that may contain the Mycobacterium tuberculosis complex. Furthermore, each 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, sonication, disruption, or grinding.

[0022] 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.

[0023] 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.

[0024] 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 test results. For example, the time from sample collection to obtaining genetic test 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 the nucleic acid purification step, the time required for nucleic acid purification can be eliminated, allowing for simple and rapid detection of Mycobacterium tuberculosis complex.

[0025] [Process (2)] In one embodiment, step (2) is preferably a step of generating nucleic acid amplification products by nucleic acid amplification, specifically, a step of generating one or more nucleic acid amplification products by performing a nucleic acid amplification reaction using one or more primers or primer sets for Mycobacterium tuberculosis complex nucleic acid amplification. 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, for example, 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, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, RPA, 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.

[0026] (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 sample-induced amplification inhibition. Furthermore, when step (3) is performed using melting curve analysis, a DNA polymerase belonging to Family B that lacks 5'->3' exonuclease activity is also preferred for use with a fluorescence-quenching probe.

[0027] 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.

[0028] (DNA polymerase) The DNA polymerase that can be used in step (2) is preferably, but 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 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] (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 Family A DNA polymerase, 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 enabling simple, rapid, and highly sensitive detection of Mycobacterium tuberculosis complex, as will be shown in the Examples below.

[0033] (Primers and primer sets for nucleic acid amplification of Mycobacterium tuberculosis complex) The primers or primer sets that can be used in step (2) are not particularly limited as long as they are capable of amplifying nucleic acid fragments derived from the Mycobacterium tuberculosis complex (particularly nucleic acid fragments that can form a complex with the probe or probe set for detecting the Mycobacterium tuberculosis complex described below). Specific examples include primers or primer sets that can amplify a nucleotide sequence containing part or all of the IS6110 region shown in SEQ ID NO: 57 and / or primers or primer sets that can amplify a nucleotide sequence containing part or all of the dnaJ gene shown in SEQ ID NO: 58. In a specific embodiment, it is preferable to use a primer or primer set that can amplify a nucleotide sequence containing part or all of the IS6110 region shown in SEQ ID NO: 57 in combination with a primer or primer set that can amplify a nucleotide sequence containing part or all of the dnaJ gene shown in SEQ ID NO: 58.

[0034] In one embodiment, a primer for amplifying nucleic acid of Mycobacterium tuberculosis complex, which is used in combination with a probe or probe set for detecting Mycobacterium tuberculosis complex described below, has any one of the following oligonucleotides (E) to (J): (E) an oligonucleotide comprising a base sequence E1 of 23 to 28 consecutive bases in the base sequence from positions 50 to 90 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence E2 which differs from the base sequence E1 by 1 to 3 bases; (F) an oligonucleotide comprising a base sequence F1 of 23 to 28 consecutive bases in the base sequence from positions 115 to 205 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence F2 which differs from the base sequence F1 by 1 to 3 bases; (G) an oligonucleotide comprising a base sequence G1 of 23 to 28 consecutive bases in the base sequence from positions 230 to 280 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence G2 which differs from the base sequence G1 by 1 to 3 bases; (H) an oligonucleotide comprising a base sequence H1 of 23 to 28 consecutive bases in the base sequence of positions 355 to 400 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence H2 which differs from said base sequence by 1 to 3 bases; (I) an oligonucleotide comprising a base sequence I1 of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence I2 which differs from said base sequence by 1 to 3 bases; (J) An oligonucleotide comprising a base sequence J1 of 19 to 25 consecutive bases in the base sequence from positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence J2 which differs from said base sequence by 1 to 3 bases.

[0035] The primers for amplifying Mycobacterium tuberculosis complex nucleic acids containing oligonucleotides (E) to (J) may be used singly, but are preferably used as a primer set consisting of two or more primers in combination. When used as a primer set, it is preferable to use a primer set designed so that one primer is complementary to the nucleic acid extension product of the other primer.

[0036] Preferred primer sets that can be used in the present invention include, for example, the primer sets shown in a) to c) below. a) a primer set for amplifying, by a nucleic acid amplification method, an oligonucleotide comprising the 79th to 130th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 57 or a nucleotide sequence complementary thereto, the primer set comprising at least one primer having an oligonucleotide (E) and at least one primer having an oligonucleotide (F); b) a primer set for amplifying, by a nucleic acid amplification method, an oligonucleotide comprising the 277th to 312th nucleotide sequence of SEQ ID NO: 57 or a nucleotide sequence complementary thereto, the primer set comprising at least one primer having an oligonucleotide (G) and at least one primer having an oligonucleotide (H); c) A primer set for amplifying an oligonucleotide comprising the 37th to 84th base sequence of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto by a nucleic acid amplification method, the primer set comprising at least one primer having an oligonucleotide (I) and at least one primer having an oligonucleotide (J).

[0037] Of the above primer sets, a) and b) are primer sets targeting the IS6110 region, and c) is a primer set targeting the dnaJ gene.

[0038] The primer set a) is preferably used in combination with a probe for detecting the Mycobacterium tuberculosis complex having the oligonucleotide (A).

[0039] The primer set b) is preferably used in combination with a probe for detecting the Mycobacterium tuberculosis complex having the oligonucleotide (B).

[0040] The primer set c) is preferably used in combination with a probe for detecting the Mycobacterium tuberculosis complex having the oligonucleotide (C).

[0041] A preferred primer set to be used when a probe set is used in the present invention can be appropriately selected depending on the type of probe included in the probe set. By using a primer set in such a combination, it is possible to more reliably detect Mycobacterium tuberculosis complex.

[0042] More specifically, preferred examples of oligonucleotide (E) include, but are not limited to, an oligonucleotide containing a base sequence shown in any one of SEQ ID NOs: 1 to 3 or a base sequence complementary thereto, or a base sequence that differs from the base sequence by 1 to 3 bases.

[0043] Preferred examples of oligonucleotide (F) include, but are not limited to, an oligonucleotide containing a base sequence shown in any one of SEQ ID NOs: 13 to 16 or a base sequence complementary thereto, or a base sequence that differs from the base sequence by 1 to 3 bases.

[0044] Preferred examples of oligonucleotide (G) include, but are not limited to, oligonucleotides containing the base sequence shown in any one of SEQ ID NOs: 4 to 12 or a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases.

[0045] Preferred examples of oligonucleotide (H) include, but are not limited to, oligonucleotides containing the base sequence shown in any of SEQ ID NOs: 17 to 22 (more preferably SEQ ID NOs: 17 to 21) or a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases.

[0046] Preferred examples of oligonucleotide (I) include, but are not limited to, oligonucleotides containing the base sequence shown in any one of SEQ ID NOs: 33 to 37 or a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases.

[0047] Preferred examples of oligonucleotide (J) include, but are not limited to, an oligonucleotide containing a base sequence shown in any one of SEQ ID NOs: 38 to 47 or a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases.

[0048] With respect to the base sequence of the primer, the "difference" in "difference of 1 to 3 bases" includes, but is not limited to, substitution, deletion, insertion, or addition of nucleotides. Substitution is preferred. Bases substituted from the original base sequence may include adenine, thymine, cytosine, guanine, as well as uracil, universal bases, and mixed bases.

[0049] The degree of "difference" is not particularly limited as long as it is capable of hybridizing with part or all of the nucleic acid derived from the tubercle bacillus complex to form a complex.

[0050] The universal base 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-mentioned ability can be used as the universal base, and preferred are inosine, deoxyinosine, and 5-nitroindole.

[0051] The mixed bases refer 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 when all of A, T, G, and C are possible, they are represented as N. This specification also follows this notation.

[0052] The number of base differences may be preferably 1 or 2, and more preferably 1. As will be shown in the results of the Examples described below, it has been demonstrated that Mycobacterium tuberculosis complex can be detected with sufficient sensitivity even when primers designed in different regions shifted by 1 to 3 bases are used.

[0053] The primers of the present invention can be used in any nucleic acid amplification method, but are preferably PCR primers used in PCR. Among them, a PCR primer set is preferred, in which one primer is designed to be complementary to the nucleic acid extension product of the other primer so that a PCR reaction can be performed.

[0054] [Process (3)] Step (3) may be performed by any method known in the art. The target M. tuberculosis complex may mutate, as with other infectious microorganisms. Mismatches between the base sequence of a primer or probe and the base sequence of a target M. tuberculosis complex mutant strain may reduce the binding strength of the primer or probe to the target gene or the nucleic acid amplification product (target nucleic acid) derived therefrom. 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. This reduces the impact of mismatches compared to real-time PCR. Therefore, it is particularly preferable to detect the nucleic acid amplification product using melting curve analysis in step (3). 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).

[0055] In one embodiment, step (3) comprises the following steps (3-1) and (3-2): (3-1) hybridizing one or more nucleic acid amplification products of step (2) with one or more probes or probe sets 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 the tubercle bacillus complex, it is preferable to use a probe or probe set described below that can specifically react with the nucleic acid amplification product derived from the tubercle bacillus complex in step (2) to form a complex.

[0056] The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of one or more nucleic acid amplification products with one or more probes or probe sets, 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 probe.

[0057] (Probe for detecting Mycobacterium tuberculosis complex) The probe for detecting the Mycobacterium tuberculosis complex of the present invention preferably contains any one of oligonucleotides (A) to (C). Use of such a nucleic acid probe enables highly sensitive detection of the Mycobacterium tuberculosis complex, for example, in melting curve analysis. The nucleic acid probe can also be used in real-time PCR and the like. Furthermore, the nucleic acid probe may be characterized by its low cross-reactivity.

[0058] The length of the probe for detecting mycobacterium tuberculosis complex of the present invention is preferably 15 to 24 bases inclusive. In one embodiment, the length of the probe for detecting mycobacterium tuberculosis complex may be, for example, 16 bases or more, preferably 17 bases or more, more preferably 18 bases or more, and even more preferably 19 bases or more. In another embodiment, the length of the probe for detecting mycobacterium tuberculosis complex may be, for example, 23 bases or less, preferably 22 bases or less, more preferably 21 bases or less, even more preferably 20 bases or less, and even more preferably 19 bases or less.

[0059] Nucleotide sequences of various strains of the M. tuberculosis complex have been analyzed, and it has been found that differences in the nucleotide sequences of various genes can be observed between strains. These differences can manifest as deletions or insertions in the nucleotide sequences, as well as single nucleotide polymorphisms.

[0060] If such differences in base sequence occur between strains in the base sequence to which the nucleic acid probe can bind, there is a risk that the reactivity of the nucleic acid probe will vary depending on the strain. This can be particularly problematic in the case of the Mycobacterium tuberculosis complex, which has a wide variety of strains. When detecting the Mycobacterium tuberculosis complex, in order to reduce the risk and suppress the influence of differences between strains, the length of the probe for detecting the Mycobacterium tuberculosis complex may be, for example, 30 bases or less, 27 bases or less, or 25 bases or less, but it is preferable to design it as a relatively short probe with a maximum length of 24 bases.

[0061] In one embodiment, the probe for detecting the Mycobacterium tuberculosis complex of the present invention may be a probe having oligonucleotide (A) or (B), wherein the length of oligonucleotide (A) or (B) is 19 to 24 bases. Use of a nucleic acid probe of such a length enables more effective detection of the Mycobacterium tuberculosis complex.

[0062] In one embodiment, the probe for detecting the Mycobacterium tuberculosis complex of the present invention may be a probe having an oligonucleotide (C) with a length of 15 to 19 bases. Use of a nucleic acid probe with such a length enables more effective detection of the Mycobacterium tuberculosis complex.

[0063] More specifically, preferred examples of oligonucleotide (A) include, but are not limited to, oligonucleotides containing the base sequence shown in any of SEQ ID NOs: 23 to 28 (more preferably SEQ ID NOs: 24 to 28) or a base sequence complementary thereto.

[0064] Preferred examples of oligonucleotide (B) include, but are not limited to, oligonucleotides containing the base sequence shown in any of SEQ ID NOs: 29 to 31 (more preferably SEQ ID NO: 30 or 31) or a base sequence complementary thereto.

[0065] Preferred examples of oligonucleotide (C) include, but are not limited to, oligonucleotides containing the base sequence shown in any of SEQ ID NOs: 48 to 56 (more preferably SEQ ID NOs: 48 to 51) or a base sequence complementary thereto.

[0066] As long as the effects of the present invention are achieved, the oligonucleotides (A) to (C) may be probes that differ from the aforementioned base sequence by 1 to 3 bases, for example, probes that differ from the aforementioned base sequence by 1 or 2 bases.

[0067] Preferably, the probe is labeled at only one of the 5'-end and 3'-end. In one embodiment, the probe for detecting Mycobacterium tuberculosis complex 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 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.

[0068] 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 interacting with guanine when hybridized with a target nucleic acid amplification product. 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).

[0069] More specifically, examples of fluorescence quenching dyes that undergo quenching through 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.

[0070] 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.

[0071] In a particularly preferred embodiment, the method for detecting the tuberculosis complex of the present invention uses a combination of two or more probes for detecting the tuberculosis complex selected from the probes for detecting the tuberculosis complex having oligonucleotides (A) to (C). By using a probe set combining two or more probes for detecting the tuberculosis complex of the present invention in this way, it is possible to detect the tuberculosis complex with high sensitivity, for example, by targeting the IS6110 region, while also being able to detect with high sensitivity strains of the tuberculosis complex that lack the IS6110 region and strains of the tuberculosis complex that have a single nucleotide polymorphism in the IS6110 region, thereby enabling more accurate detection of the tuberculosis complex.

[0072] In one preferred embodiment, the probe set for detecting Mycobacterium tuberculosis complex that can be used in the present invention preferably comprises at least one probe selected from a probe for detecting Mycobacterium tuberculosis complex having oligonucleotide (A) and a probe for detecting Mycobacterium tuberculosis complex having oligonucleotide (B), and at least one probe selected from a probe for detecting Mycobacterium tuberculosis complex having oligonucleotide (C). In one preferred embodiment, the probe set for detecting Mycobacterium tuberculosis complex may be such that oligonucleotide (A) comprises the nucleotide sequence shown in any of SEQ ID NOs: 23 to 28 or a nucleotide sequence complementary thereto, oligonucleotide (B) comprises the nucleotide sequence shown in any of SEQ ID NOs: 23 to 31 or a nucleotide sequence complementary thereto, and oligonucleotide (C) comprises the nucleotide sequence shown in any of SEQ ID NOs: 48 to 56 or a nucleotide sequence complementary thereto.

[0073] In a particularly preferred embodiment, the probe set for detecting mycobacterium tuberculosis complex that can be used in the present invention may be a probe set containing at least one probe selected from probes for detecting mycobacterium tuberculosis complex having oligonucleotide (B) and at least one probe selected from probes for detecting mycobacterium tuberculosis complex having oligonucleotide (C). The results of the test examples described below demonstrate that such a probe set enables detection of mycobacterium tuberculosis complex with higher sensitivity. For example, a suitable example of a probe set for detecting mycobacterium tuberculosis complex may be a combination of a probe having an oligonucleotide containing a nucleotide sequence represented by any one of SEQ ID NOS: 30 and 31 or a complementary nucleotide sequence thereto as the probe for detecting mycobacterium tuberculosis complex having oligonucleotide (B), and a probe having an oligonucleotide containing a nucleotide sequence represented by any one of SEQ ID NOS: 48 to 51 or a complementary nucleotide sequence thereto as the probe for detecting mycobacterium tuberculosis complex having oligonucleotide (C).

[0074] In a particularly preferred embodiment, the probe or probe set for detecting the tuberculosis complex of the present invention is used in step (3). Thus, the use of the probe or probe set of the present invention enables specific detection of the tuberculosis complex. In the method of the present invention, the probe for detecting the tuberculosis complex of the present invention may be used alone or in combination of two or more.

[0075] 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), one or more nucleic acid probes or probe sets of the present invention are hybridized to one or more nucleic acid amplification products in the reaction solution, and the fluorescence intensity of the reaction solution is measured, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) in step (2) in real time. (3-b) After completion of step (2), one or more nucleic acid probes or probe sets of the present invention are hybridized to one or more nucleic acid amplification products in the reaction solution, and the fluorescence intensity of the reaction solution is measured, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) in step (2) at an endpoint. (3-c) After completion of step (2), a step of hybridizing one or more nucleic acid probes or probe sets of the present invention to one or more nucleic acid amplification products in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution. Step (3-a), (3-b), or (3-c) allows for simple, rapid, and highly sensitive detection of the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe (including each nucleic acid probe of a probe set). 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.

[0076] (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.

[0077] (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 a target nucleic acid that may be contained in a sample can also 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.

[0078] (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 Tm values ​​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 here include single-nucleotide polymorphisms, base substitutions, base deletions, base insertions, and the like.

[0079] 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.

[0080] [Reagents for detecting Mycobacterium tuberculosis complex] In another embodiment, the present invention provides a reagent for detecting the tuberculosis complex. The reagent preferably contains at least components necessary for nucleic acid amplification reaction and detection, in addition to the probe or probe set for detecting the tuberculosis complex 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 primer set for nucleic acid amplification (particularly PCR) of tuberculosis bacteria, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. The primer set and probe may include multiple sets for amplifying multiple regions of the Mycobacterium tuberculosis complex. Preferably, the primer set and probe include at least one selected from a Mycobacterium tuberculosis complex detection probe having oligonucleotide (A) and a Mycobacterium tuberculosis complex detection probe having oligonucleotide (B), at least one selected from a Mycobacterium tuberculosis complex detection probe having oligonucleotide (C), and a primer (or primer set) capable of amplifying a region to which each of these probes can bind. More preferably, the primer set includes a combination of at least one Mycobacterium tuberculosis complex detection probe having oligonucleotide (B), at least one Mycobacterium tuberculosis complex detection probe having oligonucleotide (C), and a primer (or primer set) capable of amplifying a region to which each of these probes can bind. The concentration of each component can be adjusted as appropriate. For example, the Mycobacterium tuberculosis complex detection probe is preferably 0.01 to 1 μM, and more preferably 0.02 to 0.5 μM. When used as a probe set for detecting Mycobacterium tuberculosis complex, each probe included in the probe set is preferably within the above-mentioned concentration range. The primer for amplifying Mycobacterium tuberculosis complex nucleic acid is preferably 0.01 to 10 μM. When used as a primer set for amplifying Mycobacterium tuberculosis complex nucleic acids, each primer contained in the primer set is preferably within the above-mentioned concentration ranges. DNA polymerase is preferably 0.01 to 1 U / μL, more preferably 0.02 to 0.5 U / μL. Deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. Inorganic salts such as magnesium salts are preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0081] 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.

[0082] [Reagent kit for detecting tuberculosis complex] In another embodiment, the present invention provides a reagent kit for detecting the tuberculosis complex. The kit of the present invention is not particularly limited as long as it contains the probe or probe set for detecting the tuberculosis complex of the present invention described above, and, if necessary, further contains at least a reagent of the present invention containing a primer (or primer set) for amplifying nucleic acids of the tuberculosis complex as described above, and is configured to detect (including differentiate) the tuberculosis complex. For example, the kit of the present invention can optionally contain a reagent capable of detecting or quantifying the presence of the target tuberculosis complex, and / or instructions for use, etc. For example, the kit of the present invention can be provided in a form in which the 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, for example, in a single package, and information on how to use the kit is included. The kit of the present invention can also contain a positive control solution and / or a negative control solution. [Example]

[0083] The present invention will be described in detail below based on examples, but the present invention is not limited to the following examples.

[0084] Example 1: Detection and confirmation of Mycobacterium tuberculosis complex DNA (1) Method To confirm whether the probe containing oligonucleotide (A) can detect Mycobacterium tuberculosis complex DNA, primers were used to amplify Mycobacterium tuberculosis complex DNA (1 copy / μL), and then a probe containing oligonucleotide (A) was bound to the resulting nucleic acid amplification product, followed by a melting curve to detect the nucleic acid amplification product derived from Mycobacterium tuberculosis complex DNA.

[0085] (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 shown in SEQ ID NO: 1 3.0 μM primer shown in SEQ ID NO: 16 0.4 μM of the probe shown by SEQ ID NO: 25 (BODIPY-FL labeled at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0086] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0087] (4) Results Figure 1 shows the results of changes in fluorescence intensity detected in melting curve analysis, with the horizontal axis representing temperature and the vertical axis representing the differential fluorescence signal. The probe in this example is a quenching probe, characterized in that its terminus is labeled with a fluorescent substance. When it binds to an oligonucleotide having a complementary base sequence to form a complex, the fluorescent substance is quenched by proximity to guanine, and then emits light when the probe is released from the oligonucleotide and becomes independent. Therefore, if a nucleic acid amplification product derived from M. tuberculosis DNA is obtained by nucleic acid amplification and the probe is capable of detecting the nucleic acid amplification product derived from M. tuberculosis DNA, the nucleic acid amplification product and the probe form a complex during the 30-second incubation step at 39°C in the melting curve analysis. Then, during the subsequent heating step from 40°C to 75°C, the probe releases from the nucleic acid amplification product and emits light at a specific temperature. Therefore, changes in fluorescence intensity are detected during the heating process, and the amount of change in fluorescence appears as a peak on the graph. As clear peaks are observed in Figure 1, it was demonstrated that nucleic acid amplification products derived from Mycobacterium tuberculosis DNA can be detected with high specificity by generating nucleic acid amplification products using Mycobacterium tuberculosis DNA as a sample using the primer set shown in SEQ ID NO: 1 and 16 and detecting them with the probe shown in SEQ ID NO: 25. Furthermore, the measurement time in this example was approximately 30 minutes, enabling rapid detection of tuberculosis DNA.

[0088] Example 2: Primer-probe combination test (1) Method To confirm whether a probe containing oligonucleotide (B) could also specifically detect M. tuberculosis complex DNA, we performed nucleic acid amplification of M. tuberculosis complex DNA (1 copy / μL) using primers, then bound the probe containing oligonucleotide (B) to the resulting nucleic acid amplification product, performed a melting curve, and attempted to detect the nucleic acid amplification product derived from M. tuberculosis complex DNA. Note that, to confirm reproducibility in this experiment, measurements were performed with N=4 for each primer-probe combination.

[0089] (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 shown in either SEQ ID NO: 4 or 12 0.3 μM Primer shown in any one of SEQ ID NOs: 17, 18, and 22 0.3 μM probe shown in either SEQ ID NO: 29 or 30 (BODIPY-FL labeled at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0090] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0091] (4) Results Table 1 shows the primer-probe combinations confirmed in this example and the average fluorescence change when those combinations were used to detect M. tuberculosis DNA. As shown in these results, M. tuberculosis DNA was detected in all combinations and all measurements by using a probe containing oligonucleotide (B). Of the primers used in this example, the primer set shown in SEQ ID NOs: 12 and 22 is described in Non-Patent Document 2 as being used in combination with a TaqMan probe (SEQ ID NO: 32) designed in a region different from that of the present invention, and these primers are capable of binding to the IS6110 region and amplifying nucleic acids, like the other primers shown in SEQ ID NOs: 4, 17, and 18. In this example, the combination of the primer set shown in SEQ ID NOs: 12 and 22 with the probe shown in SEQ ID NO: 29 also enabled detection of Mycobacterium tuberculosis DNA with sufficient fluorescence intensity, confirming the effectiveness of this combination with a probe having oligonucleotide (B) of the present invention. On the other hand, it was confirmed that the combination of the probe of the present invention with other primer sets (particularly, a primer set in which at least one primer is SEQ ID NO: 17 or 18, particularly a primer set consisting of a primer shown in SEQ ID NO: 12 and a primer shown in SEQ ID NO: 17 or 18) resulted in a higher fluorescence change and was superior to the primer set shown in SEQ ID NO: 12 and 22. Because the fluorescence change correlates to some extent with the amount of nucleic acid amplification product, such specific primer set combinations are considered to be particularly excellent in DNA amplification ability.

[0092] [Table 1]

[0093] Example 3: Primer-probe combination test (1) Method To confirm whether a probe containing oligonucleotide (C) could also specifically detect M. tuberculosis complex DNA, M. tuberculosis complex DNA (50 copies / μL) was amplified using primers, and then a probe containing oligonucleotide (C) was bound to the resulting nucleic acid amplification product, followed by a melting curve to attempt to detect the nucleic acid amplification product derived from M. tuberculosis complex DNA.

[0094] (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. 2.5 μM of a primer represented by any one of SEQ ID NOs: 35 to 37 0.5 μM of a primer represented by any one of SEQ ID NOs: 41 to 43 0.25 μM of a probe represented by any one of SEQ ID NOs: 49 to 51 (BODIPY-FL labeled at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0095] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0096] (4) Results Table 2 shows the primer-probe combinations confirmed in this example and the fluorescence change observed when these combinations were used to detect M. tuberculosis DNA. If the fluorescence change exceeds 4 and a peak shape is observed in the melting curve analysis results, it can be determined that M. tuberculosis DNA has been detected. In this example, Mycobacterium tuberculosis DNA was detected in all combinations and all measurements using a probe containing oligonucleotide (C). In particular, it was confirmed that the use of a primer set in which at least one primer is SEQ ID NO: 41 or 42 in combination with a probe containing oligonucleotide (C) tends to produce a stable and high change in fluorescence.

[0097] [Table 2]

[0098] Example 4: Primer-probe combination test (1) Method To further confirm that the probe of the present invention is effective in detecting Mycobacterium tuberculosis complex DNA, Mycobacterium tuberculosis complex DNA (2 copies / μL) was amplified using primers, and then the probe of the present invention was bound to the resulting nucleic acid amplification product, followed by a melting curve analysis to detect the nucleic acid amplification product derived from Mycobacterium tuberculosis complex DNA.

[0099] (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. 2.5 μM of a primer represented by any one of SEQ ID NOs: 6 to 8 0.5 μM primer shown in SEQ ID NO: 20 0.25 μM of a probe represented by any one of SEQ ID NOs: 29 to 31 (BODIPY-FL labeled at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0100] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0101] (4) Results Table 3 shows the primer-probe combinations confirmed in this example and the fluorescence change observed when these combinations were used to detect M. tuberculosis DNA. If the fluorescence change exceeds 4 and a peak shape is observed in the melting curve analysis results, it can be determined that M. tuberculosis DNA has been detected. In this example, evaluation was performed using a single fixed primer 2 (reverse primer), three combinations of primers 1 (forward primers), and three probes (including the probe of SEQ ID NO: 30) containing oligonucleotide (B), which was confirmed to exhibit high detection ability for the M. tuberculosis complex in Example 2. The results confirmed that M. tuberculosis DNA could be detected with high sensitivity in all combinations and all measurements. These results also indicated that the combination of a primer represented by any of SEQ ID NOs: 6 to 8 with the primer represented by SEQ ID NO: 20 was suitable. Furthermore, it was revealed that in addition to the probe represented by SEQ ID NO: 30, the probes represented by SEQ ID NOs: 29 and 31, each of which differs from the probe by approximately one base, could also detect the M. tuberculosis complex with high sensitivity.

[0102] [Table 3]

[0103] Example 5: Primer-probe combination test (1) Method To further confirm that the probe of the present invention is effective in detecting Mycobacterium tuberculosis complex DNA, Mycobacterium tuberculosis complex DNA (2 copies / μL) was amplified using primers, and then the probe of the present invention was bound to the resulting nucleic acid amplification product, followed by a melting curve analysis to detect the nucleic acid amplification product derived from Mycobacterium tuberculosis complex DNA.

[0104] (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 of a primer shown in any one of SEQ ID NOs: 1 to 3 2.5 μM of a primer represented by any one of SEQ ID NOs: 13 to 16 0.25 μM probe shown in either SEQ ID NO: 24 or 26 (BODIPY-FL labeled at the 3' end), or Probe represented by SEQ ID NO: 28 (BODIPY-FL labeled at the 5' end, phosphorylated at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0105] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0106] (4) Results Table 4 shows the primer-probe combinations confirmed in this example and the amount of change in fluorescence when these combinations were used to detect Mycobacterium tuberculosis DNA. In this example, evaluation was carried out using various oligonucleotide (A) probes, including those differing by one or several bases from the probe shown in SEQ ID NO: 25, the effectiveness of which was confirmed in Example 1. As a result, it was confirmed that Mycobacterium tuberculosis DNA was detected with a large change in fluorescence in all combinations of various primers and probes that amplify regions to which these probes can bind.

[0107] [Table 4]

[0108] Example 6: Primer-probe combination test (1) Method To further confirm that the probe of the present invention is effective in detecting Mycobacterium tuberculosis complex DNA, Mycobacterium tuberculosis complex DNA (2 copies / μL) was amplified using various primers, and then the probe of the present invention was bound to the resulting nucleic acid amplification product, followed by a melting curve analysis to detect the nucleic acid amplification product derived from Mycobacterium tuberculosis DNA.

[0109] (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. 2.5 μM primers shown in either SEQ ID NO: 8 or 10 0.5 μM of a primer represented by any one of SEQ ID NOs: 19 to 21 0.25 μM probe shown in either SEQ ID NO: 30 or 31 (BODIPY-FL labeled at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0110] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0111] (4) Results Table 5 shows the primer-probe combinations confirmed in this example and the amount of change in fluorescence when these combinations were used to detect Mycobacterium tuberculosis DNA. In this example, the probe shown in SEQ ID NO: 30 or 31, which was confirmed to exhibit high ability to detect the M. tuberculosis complex in Examples 2 and 4 above, was evaluated in combination with various primers. As a result, it was confirmed that M. tuberculosis DNA was detected with a large change in fluorescence in all combinations of various primer sets, such as the combination of the primer shown in SEQ ID NO: 20 with the primer shown in SEQ ID NO: 19 or 21, which has several base differences at each of the 5' and 3' ends, and the primer shown in SEQ ID NO: 8 or 10.

[0112] [Table 5]

[0113] Example 7: Primer-probe combination test (1) Method To further confirm that the probe of the present invention is effective in detecting Mycobacterium tuberculosis complex DNA, Mycobacterium tuberculosis complex DNA (50 copies / μL) was amplified using various primers, and then the probe of the present invention was bound to the resulting nucleic acid amplification product, followed by a melting curve analysis to detect the nucleic acid amplification product derived from Mycobacterium tuberculosis DNA.

[0114] (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. 2.5 μM of a primer represented by any one of SEQ ID NOs: 35 to 37 0.5 μM Primer shown in any one of SEQ ID NOs: 39, 45, 46, and 47 0.25 μM probe shown in either SEQ ID NO: 48 or 49 (BODIPY-FL labeled at the 3' end) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0115] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0116] (4) Results Table 6 shows the primer-probe combinations confirmed in this example and the amount of change in fluorescence when these combinations were used to detect Mycobacterium tuberculosis DNA. In this example, the probe shown in SEQ ID NO: 48, which was confirmed to exhibit high ability to detect the Mycobacterium tuberculosis complex in Example 3 above, and the probe shown in SEQ ID NO: 49, which differs from the probe by one nucleotide, were used in combination with various primers for evaluation. As a result, it was confirmed that when the primer shown in SEQ ID NO: 36 was combined with the primer shown in SEQ ID NO: 46, detection of Mycobacterium tuberculosis complex DNA was possible, but the amount of fluorescence change was somewhat low. However, it was confirmed that all other combinations detected Mycobacterium tuberculosis DNA with a high amount of fluorescence change.

[0117] [Table 6]

[0118] Example 8: Combination test of probe sets (1) Method To confirm the effect on the detection of M. tuberculosis complex DNA when two or more probes containing any of the oligonucleotides (A) to (C) were mixed, two types of primers and one type of probe were used as one set. Two different sets of primers and probes were used to amplify M. tuberculosis complex DNA (2 copies / μL) and detect the nucleic acid amplification products.

[0119] (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. A μM Primer shown in any of SEQ ID NOs: 2, 6, and 10 BμM Primer represented by any one of SEQ ID NOs: 14, 19, and 20 0.25 μM probes shown in any of SEQ ID NOs: 26, 30, and 31 (BODIPY-FL labeled at the 3' end) 2.5 μM of a primer represented by any one of SEQ ID NOs: 35 to 37 0.5 μM Primer shown in any one of SEQ ID NOs: 39, 42, and 43 0.25 μM probes represented by any of SEQ ID NOs: 48, 49, and 51 (BODIPY-FL labeled at the 3' end) (In the above reaction solution composition, A μL was 0.5 μL for SEQ ID NO: 2 and 2.5 μL for SEQ ID NO: 6 or 10; B μL was 2.5 μL for SEQ ID NO: 14 and 0.5 μL for SEQ ID NO: 19 or 20.) The reaction solution was mixed with 4 μL of M. tuberculosis DNA.

[0120] (3) Reaction Using GENECUBE (registered trademark), PCR was carried out by reacting the reaction solution through the following temperature cycles. 94℃ 30 seconds, 97℃ 1 sec - 58℃ 3 sec - 63℃ 5 sec (50 cycles) After PCR, the reaction was carried out at the following temperatures and a melting curve analysis was performed. 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (temperature rises at 0.09℃ / sec)

[0121] (4) Results Table 7 shows the combinations of probe sets and primers confirmed in this example, and the amount of change in fluorescence when tuberculosis DNA was detected using those combinations. In this example, a probe set combining a probe containing oligonucleotide (A) or (B) that detects the IS6110 region of M. tuberculosis complex with a probe containing oligonucleotide (C) that detects the dnaJ gene of M. tuberculosis complex was evaluated using a primer set that amplifies the region to which each probe can bind. The results confirmed that M. tuberculosis DNA was detected in all combinations and all measurements. In particular, when the probe containing oligonucleotide (B) and the probe containing oligonucleotide (C) were used in combination, the amount of fluorescence change was greater than when each probe was used alone, enabling clearer DNA detection. From the above, it was confirmed that the use of multiple probes of the present invention in combination as a set can be used to detect Mycobacterium tuberculosis complex DNA, and that it is particularly preferable to use a combination of a probe having oligonucleotide (B) and a probe having oligonucleotide (C).

[0122] [Table 7] [Industrial Applicability]

[0123] The use of the primers and probes described in the present invention enables specific detection of Mycobacterium tuberculosis complex DNA, which is believed to enable rapid and reliable detection of Mycobacterium tuberculosis complex, leading to early diagnosis and treatment of tuberculosis.

Claims

1. A probe for detecting Mycobacterium tuberculosis complex, comprising any one of the following oligonucleotides (A) to (C): (A) an oligonucleotide comprising a base sequence of 15 to 24 consecutive bases in the base sequence of positions 79 to 130 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence differing from said base sequence by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye; (B) an oligonucleotide comprising a base sequence of 15 to 24 consecutive bases in the base sequence from 277th to 312th of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence differing from said base sequence by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye; (C) An oligonucleotide comprising a base sequence of 15 to 24 consecutive bases in the base sequence from 37 to 84 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases, wherein at least one of the terminal bases at the 5' end and the 3' end is cytosine, and the cytosine at one of the terminal bases at the 5' end and the 3' end is labeled with a fluorescent dye.

2. 2. The probe for detecting a Mycobacterium tuberculosis complex according to claim 1, wherein the length of each of the oligonucleotides (A) to (C) is 19 to 24 bases.

3. 2. The probe for detecting a Mycobacterium tuberculosis complex according to claim 1, wherein the length of each of the oligonucleotides (A) to (C) is 15 to 19 bases.

4. the oligonucleotide (A) comprises a base sequence shown in any one of SEQ ID NOs: 23 to 28 or a base sequence complementary thereto; the oligonucleotide (B) comprises a base sequence shown in any one of SEQ ID NOs: 29 to 31 or a base sequence complementary thereto; The probe for detecting a Mycobacterium tuberculosis complex according to claim 1, wherein the oligonucleotide (C) comprises a base sequence represented by any one of SEQ ID NOs: 48 to 56.

5. 2. The probe for detecting Mycobacterium tuberculosis complex according to claim 1, wherein the fluorescent dye is a fluorescence quenching dye that is quenched by interaction with guanine.

6. 2. The probe for detecting Mycobacterium tuberculosis complex according to claim 1, wherein the fluorescent dye is 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.

7. The probe for detecting Mycobacterium tuberculosis complex according to claim 1, wherein the fluorescent dye is 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).

8. A probe set for detecting the tubercle bacillus complex, comprising at least two probes selected from the probes for detecting the tubercle bacillus complex having the oligonucleotides (A) to (C) according to claim 1.

9. 9. The tuberculosis complex detection probe set according to claim 8, comprising at least one probe selected from a tuberculosis complex detection probe having the oligonucleotide (A) and a tuberculosis complex detection probe having the characteristics of the oligonucleotide (B), and at least one probe selected from a tuberculosis complex detection probe having the oligonucleotide (C).

10. The probe set for detecting tuberculosis complex according to claim 9, comprising at least one probe selected from probes for detecting tuberculosis complex having the oligonucleotide (B) and at least one probe selected from probes for detecting tuberculosis complex having the oligonucleotide (C).

11. A primer for amplifying nucleic acid of the Mycobacterium tuberculosis complex, which is used in combination with the probe for detecting the Mycobacterium tuberculosis complex according to any one of claims 1 to 7 or the probe set for detecting the Mycobacterium tuberculosis complex according to any one of claims 8 to 10, and which comprises any one of the following oligonucleotides (E) to (J): (E) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 50 to 90 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (F) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 115 to 205 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from said base sequence; (G) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 230 to 280 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (H) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 355 to 400 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases.

12. the oligonucleotide (E) comprises a base sequence represented by any one of SEQ ID NOs: 1 to 3, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; the oligonucleotide (F) comprises a base sequence represented by any one of SEQ ID NOs: 13 to 16, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; the oligonucleotide (G) comprises a base sequence represented by any one of SEQ ID NOs: 4 to 12, a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases; the oligonucleotide (H) comprises a base sequence shown in any one of SEQ ID NOs: 17 to 22, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; the oligonucleotide (I) comprises a base sequence represented by any one of SEQ ID NOs: 33 to 37, a base sequence complementary thereto, or a base sequence having 1 to 3 base differences from the base sequence; The primer for amplifying Mycobacterium tuberculosis complex nucleic acid according to claim 11, wherein the oligonucleotide (J) comprises a base sequence shown in any one of SEQ ID NOs: 38 to 47, a base sequence complementary thereto, or a base sequence differing from said base sequence by 1 to 3 bases.

13. The primer for amplifying a Mycobacterium tuberculosis complex nucleic acid according to claim 11, which is a PCR primer.

14. A primer set for amplifying Mycobacterium tuberculosis complex nucleic acid, which is used in combination with the probe for detecting Mycobacterium tuberculosis complex according to any one of claims 1 to 7 or the probe set for detecting Mycobacterium tuberculosis complex according to any one of claims 8 to 10, and which comprises at least one combination selected from the group consisting of a combination of a primer having the following oligonucleotide (E) and a primer having the following oligonucleotide (F); a combination of a primer having the following oligonucleotide (G) and a primer having the following oligonucleotide (H); and a combination of a primer having the following oligonucleotide (I) and a primer having the following oligonucleotide (J): (E) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 50 to 90 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (F) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 115 to 205 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (G) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 230 to 280 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (H) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 355 to 400 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases.

15. The following steps (1), (2), and (3): (1) providing a sample that may contain the Mycobacterium tuberculosis complex; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting the one or more nucleic acid amplification products of step (2) using one or more probes or probe sets; A method for detecting a tuberculosis complex, comprising: a) detecting a tuberculosis complex; and b) detecting a tuberculosis complex using a probe or probe set; wherein the probe or probe set is a probe for detecting a tuberculosis complex according to any one of claims 1 to 7 or a probe set for detecting a tuberculosis complex according to any one of claims 8 to 10.

16. 16. The detection 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.

17. The detection method according to claim 16, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a mutant thereof.

18. The method of claim 15, wherein the nucleic acid amplification reaction in step (2) uses a primer set for amplifying Mycobacterium tuberculosis complex nucleic acid, the primer set comprising at least one combination selected from the group consisting of a combination of a primer having the following oligonucleotide (E) and a primer having the following oligonucleotide (F); a combination of a primer having the following oligonucleotide (G) and a primer having the following oligonucleotide (H); and a combination of a primer having the following oligonucleotide (I) and a primer having the following oligonucleotide (J): (E) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 50 to 90 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (F) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 115 to 205 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (G) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 230 to 280 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (H) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 355 to 400 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases.

19. A reagent for detecting the tuberculosis complex, comprising the probe for detecting the tuberculosis complex according to any one of claims 1 to 7 or the probe set for detecting the tuberculosis complex according to any one of claims 8 to 10.

20. A kit for detecting the tuberculosis complex, comprising the reagent for detecting the tuberculosis complex according to claim 19.

21. The kit for detecting the Mycobacterium tuberculosis complex according to claim 20, further comprising primers for amplifying nucleic acids of the Mycobacterium tuberculosis complex and a DNA polymerase.

22. The kit for detecting Mycobacterium tuberculosis complex according to claim 21, wherein the primers for amplifying Mycobacterium tuberculosis complex nucleic acids comprise at least one combination selected from the group consisting of a combination of a primer having the following oligonucleotide (E) and a primer having the following oligonucleotide (F); a combination of a primer having the following oligonucleotide (G) and a primer having the following oligonucleotide (H); and a combination of a primer having the following oligonucleotide (I) and a primer having the following oligonucleotide (J): (E) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 50 to 90 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (F) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 115 to 205 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases; (G) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence from positions 230 to 280 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (H) an oligonucleotide comprising a base sequence of 23 to 28 consecutive bases in the base sequence of positions 355 to 400 of the base sequence shown in SEQ ID NO: 57 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (I) an oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 1 to 40 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence having 1 to 3 base differences from said base sequence; (J) An oligonucleotide comprising a base sequence of 19 to 25 consecutive bases in the base sequence of positions 84 to 150 of the base sequence shown in SEQ ID NO: 58 or a base sequence complementary thereto, or comprising a base sequence which differs from said base sequence by 1 to 3 bases.

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