A method for identifying single-nucleotide mutations in the erm(41) gene of acid-fast bacteria belonging to the Mycobacteroides abscessus complex, and a primer set and probe for use in the method

Abstract

One or more embodiments of the present invention provides a novel means for determining a single base mutation of erm (41) gene of acid-fast bacillus belonging to Mycobacteroides abscessus complex.　One or more embodiments relate to a method for determining a base corresponding to position 28 of erm (41) gene of acid-fast bacillus belonging to Mycobacteroides abscessus complex, the method including detection of an index base sequence in the genome DNA of acid-fast bacillus which is a subject for determination, where the index base sequence is present in a region of the genome DNA other than the erm (41) gene when the base of the erm (41) gene is cytosine, and is not present when the base is thymine, thereby establishing that the presence of the index base sequence indicates that the base is cytosine, and the absence of the index base sequence indicates that the base is thymine.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a single-nucleotide mutation in the erm(41) gene of acid-fast bacteria belonging to the Mycobacteroides abscessus complex, a primer set and a probe used in the method, and a method for detecting a single-nucleotide mutation in the erm(41) gene of acid-fast bacteria, a primer set and a probe used in the method. [Background technology]

[0002] Mycobacteria are acid-fast, non-motile, gram-positive rods. Mycobacteria are broadly divided into two groups: the Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM). To date, more than 170 species of nontuberculous mycobacteria have been reported, of which approximately 30 species are known to be pathogenic to humans.

[0003] Among nontuberculous mycobacterial infections, Mycobacteroides abscessus complex infections are resistant to existing antibiotics and no effective treatment has been established, resulting in an increasing number of patients and a rise in the number of severe cases. The causative bacterial group, Mycobacteroides abscessus complex, includes three subspecies: Mycobacteroides abscessus subsp. abscessus, Mycobacteroides abscessus subsp. massiliense, and Mycobacteroides abscessus subsp. bolletii. Mycobacteroides abscessus is sometimes referred to as Mycobacterium abscessus.

[0004] Patent Document 1 discloses a primer set for distinguishing the above-mentioned three subspecies of Mycobacteroides abscessus complex, which includes a first primer that targets the downstream region of a membrane transporter gene common to the three subspecies and is designed to produce an amplified product of different sizes for each subspecies, and a second primer that targets the downstream region of an ATP-binding cassette transporter gene common to the three subspecies and is designed to produce an amplified product of different sizes for each subspecies.

[0005] It is known that Mycobacteroides abscessus complex bacteria develop resistance to antibiotics due to genetic mutations. There are macrolide-susceptible strains of Mycobacteroides abscessus complex, as well as inducible-resistant strains that acquire resistance during drug administration. The presence or absence of this inducible resistance is related to a single nucleotide polymorphism in the erm(41) gene. The T28 strain, in which the 28th base of the erm(41) gene is a thymine, is resistant to antibiotics, whereas the C28 strain, in which the 28th base is a cytosine, is susceptible (see Non-Patent Document 1).

[0006] On the other hand, Patent Document 2 describes a method for detecting mutants of bacteria such as Mycoplasma pneumoniae that are resistant to macrolide antibiotics, in which a hydrolysis probe that behaves differently between a standard strain and a mutant strain is contacted with a biological sample of a subject and then the probe is detected by real-time PCR.

[0007] A known technique for distinguishing genetic single nucleotide polymorphisms is the use of primer extension reactions (including nucleic acid amplification methods) using allele-specific primers. An allele-specific primer is a primer whose primer extension reaction efficiency varies significantly depending on the base type of the targeted single nucleotide polymorphism. Therefore, for example, PCR can be performed using an allele-specific primer and the amount of the amplified product can be analyzed to identify the base type of the targeted single nucleotide polymorphism. However, in this case, false positives can occur if the reaction conditions are not strictly defined. Patent Document 3 describes a method for distinguishing single nucleotide polymorphisms with reduced false positives, using an allele-specific primer whose 3'-terminal base corresponds to the targeted single nucleotide polymorphism, with a specific pattern for the second and third bases from the 3'-end. However, in either case, designing an allele-specific primer for directly detecting a single nucleotide polymorphism requires locating a site corresponding to the single nucleotide polymorphism near the 3'-end of the primer, which limits design flexibility. Depending on the sequence surrounding the single nucleotide polymorphism, it can be difficult to select a primer that combines sufficient primer extension efficiency for discrimination with reduced false positives. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 2019-97493 [Patent Document 2] International Publication WO2013 / 136818 [Patent Document 3] Patent No. 3859678 [Non-patent literature]

[0009] [Non-Patent Document 1] J Antimicrob Chemother 2017;72:1669-1677 Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, a single nucleotide polymorphism in the erm(41) gene is involved in the presence or absence of macrolide-induced resistance in M. abscessus complex strains. The T28 strain, which has a thymine at position 28 of the erm(41) gene, is resistant to macrolides, whereas the C28 strain, which has a cytosine at position 28, is susceptible. Therefore, distinguishing between T28 and C28 strains is useful for estimating macrolide susceptibility in M. abscessus complex strains.

[0011] To obtain allele-specific primers for directly detecting single nucleotide polymorphisms, it is necessary to design primers from a limited number of sequence candidates that have both sufficient primer extension efficiency for discrimination and low false positive reactions, as described in Patent Document 3, which is not easy to implement.

[0012] Thus, one or more embodiments of the present invention provide a novel means for determining whether the 28th base of the erm(41) gene in mycobacteria belonging to the Mycobacteroides abscessus complex is cytosine or thymine. Also, one or more embodiments of the present invention provide a novel means for determining whether the 28th base of the erm(41) gene in mycobacteria is cytosine. [Means for solving the problem]

[0013] The present inventors have surprisingly found an indicator base sequence in the genomic DNA of acid-fast bacteria belonging to the Mycobacteroides abscessus complex that is present when the 28th base of the erm(41) gene is cytosine but absent when the 28th base is thymine. They have also found that the 28th base of the erm(41) gene of the acid-fast bacterium to be evaluated can be determined based on the presence or absence of this indicator base sequence, and have completed the following invention. [1] 1. A method for determining whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine, comprising: An indicator base sequence is detected in the genomic DNA of the acid-fast bacterium to be evaluated, the indicator base sequence being present in a region of the genomic DNA other than the erm(41) gene when the base in the erm(41) gene is cytosine, and not present when the base is thymine. Including, detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine; The absence of detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is thymine. method. [2] The method according to [1], wherein the index base sequence is a first base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 2, or a second base sequence complementary to the first base sequence. [3] The method according to [2], wherein the first base sequence contains, at least in part, a base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11. [4] A method for determining the susceptibility of mycobacteria belonging to the Mycobacteroides abscessus complex to macrolide antibiotics, comprising the method according to any one of [1] to [3], detection of the indicator base sequence indicates that the acid-fast bacterium is susceptible to a macrolide antibiotic; Absence of detection of the indicator base sequence indicates that the acid-fast bacterium is not susceptible to macrolide antibiotics. The method. [5] A primer set for detecting an indicator base sequence that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is a cytosine, and is absent when the base is a thymine, a first primer containing a polynucleotide containing, at its 3' end, a base sequence f12 that can hybridize with a complementary base sequence of a partial base sequence f11 of 10 or more consecutive bases contained in the index base sequence; a second primer containing a polynucleotide containing, at its 3' end, a base sequence r12 that is contained in the index base sequence and is located closer to the 3' end than the 3' end of the partial base sequence f11 and that is capable of hybridizing to a partial base sequence r11 of 10 or more consecutive bases; A primer set comprising: [6] The primer set according to [5], wherein the index base sequence is a third base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 2. [7] The primer set according to [6], wherein the base sequence from the 5'-end base of the partial base sequence f11 to the 3'-end base of the partial base sequence r11 contained in the third base sequence contains at least a portion of a base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11. [8] The partial base sequence f11 is (f11-12-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2; (f11-18-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2; (f11-20-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2; (f11-22-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2; (f11-24-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2; (f11-26-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2, or (f11-28-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 and The partial base sequence r11 is (r11-13-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2, (r11-19-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2, (r11-21-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2, (r11-23-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2, (r11-25-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2, (r11-27-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2, or (r11-29-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 That is, The primer set according to [6] or [7]. [9] The primer set according to any one of [5] to [8], wherein one of the first primer and the second primer further comprises a labeling moiety that is a tag or a labeling substance that can be bound to a labeling substance, and the other primer further comprises a binding moiety that is a tag that can be bound to a solid phase carrier.

[10] [9] The primer set according to [9], and a solid phase carrier at least partially including a portion capable of binding to the binding portion. A kit for detecting an indicator base sequence that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of acid-fast bacteria belonging to the Mycobacteroides abscessus complex is a cytosine, but is not present when the base is a thymine.

[11] A probe for detecting an indicator base sequence that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is a cytosine, and is absent when the base is a thymine, a probe comprising a polynucleotide containing a base sequence capable of hybridizing with a partial base sequence p1 of 10 or more consecutive bases contained in the index base sequence;

[12] The probe according to

[11] , wherein the index base sequence is a first base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 2, or a second base sequence complementary to the first base sequence.

[13] The probe according to

[12] , wherein the partial base sequence p1 at least partially comprises a base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11 or a complementary base sequence thereof.

[14] The partial base sequence p1 is (p1-12-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-18-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-20-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-22-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-24-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-26-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-28-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-13-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-19-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-21-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-23-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-25-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-27-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2 or its complementary base sequence, or (p1-29-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 or its complementary base sequence The probe according to

[12] or

[13] ,

[15] 1. A method for determining whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine, comprising: carrying out a nucleic acid amplification reaction using the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined as a template and the primer set according to any one of [5] to [9]; Detecting an amplification product from the nucleic acid amplification reaction; and determining that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine when the amplification product is detected, and determining that the base is thymine when the amplification product is not detected; A method comprising:

[16] A method for determining the susceptibility of mycobacteria belonging to the Mycobacteroides abscessus complex to macrolide antibiotics, comprising the method described in

[15] , determining that the acid-fast bacterium is susceptible to macrolide antibiotics when the amplification product is detected, and determining that the acid-fast bacterium is not susceptible to macrolide antibiotics when the amplification product is not detected; A method comprising:

[17] 1. A method for determining whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine, comprising: Incubating the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined with the probe according to any one of

[11] to

[14] under conditions allowing hybridization; detecting hybridization of the genomic DNA or the polynucleotide with the probe; and determining that the base in the erm(41) gene in the genomic DNA of the acid-fast bacterium to be determined is cytosine when the hybridization is detected, and determining that the base is thymine when the hybridization is not detected; A method comprising:

[18]

[17] A method for determining the susceptibility of mycobacteria belonging to the Mycobacteroides abscessus complex to macrolide antibiotics, comprising the method described in

[17] , If the hybridization is detected, determining that the mycobacterium is susceptible to macrolide antibiotics, and if the hybridization is not detected, determining that the mycobacterium is not susceptible to macrolide antibiotics.

[19] 1. A method for determining that the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in an acid-fast bacterium, in an erm(41) gene, is cytosine, comprising: An indicator base sequence is detected in the genomic DNA of the acid-fast bacterium to be evaluated, the indicator base sequence being present in a region of the genomic DNA other than the erm(41) gene when the base in the erm(41) gene is cytosine, and not present when the base is thymine. Including, Detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium to be determined is cytosine. method.

[20] The method according to

[19] , wherein the index base sequence is a first base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 2, or a second base sequence complementary to the first base sequence. [twenty one] The method according to

[20] , wherein the first base sequence contains, at least in part, a base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11. [twenty two] A method for determining the susceptibility of mycobacteria to macrolide antibiotics, comprising the method according to any one of

[19] to

[21] , detection of the indicator base sequence indicates that the acid-fast bacterium is susceptible to a macrolide antibiotic; The method. [twenty three] A primer set for detecting an indicator base sequence that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in an acid-fast bacterium is a cytosine, and is absent when the base is a thymine, a first primer containing a polynucleotide containing, at its 3' end, a base sequence f12 that can hybridize with a complementary base sequence of a partial base sequence f11 of 10 or more consecutive bases contained in the index base sequence; a second primer containing a polynucleotide containing, at its 3' end, a base sequence r12 that is contained in the index base sequence and is located closer to the 3' end than the 3' end of the partial base sequence f11 and that is capable of hybridizing to a partial base sequence r11 of 10 or more consecutive bases; A primer set comprising: [twenty four] The primer set according to

[23] , wherein the index base sequence is a third base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 2. [twenty five] The primer set according to

[24] , wherein the base sequence from the 5'-end base of the partial base sequence f11 to the 3'-end base of the partial base sequence r11 contained in the third base sequence contains at least a portion of a base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11.

[26] The partial base sequence f11 is (f11-12-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2; (f11-18-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2; (f11-20-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2; (f11-22-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2; (f11-24-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2; (f11-26-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2, or (f11-28-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 and The partial base sequence r11 is (r11-13-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2, (r11-19-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2, (r11-21-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2, (r11-23-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2, (r11-25-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2, (r11-27-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2, or (r11-29-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 That is, The primer set according to

[24] or

[25] .

[27] The primer set according to any one of

[23] to

[26] , wherein one of the first primer and the second primer further comprises a labeling moiety that is a tag or a labeling substance that can be bound to a labeling substance, and the other of the first primer and the second primer further comprises a binding moiety that is a tag that can be bound to a solid phase carrier.

[28]

[27] A primer set according to

[27] ; and a solid phase carrier at least partially including a portion capable of binding to the binding portion. A kit for detecting an indicator base sequence in acid-fast bacteria that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene is cytosine, but is not present when the base is thymine.

[29] A probe for detecting an indicator base sequence in an acid-fast bacterium, which is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene is cytosine, and is absent when the base is thymine, a probe comprising a polynucleotide containing a base sequence capable of hybridizing with a partial base sequence p1 of 10 or more consecutive bases contained in the index base sequence;

[30] The probe according to

[29] , wherein the index base sequence is a first base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 2, or a second base sequence complementary to the first base sequence.

[31] The probe according to

[30] , wherein the partial base sequence p1 contains at least a portion of a base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11 or a complementary base sequence thereof.

[32] The partial base sequence p1 is (p1-12-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-18-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-20-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-22-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-24-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-26-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-28-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-13-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-19-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-21-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-23-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-25-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-27-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2 or its complementary base sequence, or (p1-29-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 or its complementary base sequence The probe according to

[30] or

[31] ,

[33] 1. A method for determining that the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in an acid-fast bacterium, in an erm(41) gene, is cytosine, comprising: carrying out a nucleic acid amplification reaction using the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined as a template and the primer set according to any one of

[23] to

[27] ; Detecting an amplification product from the nucleic acid amplification reaction; and determining that the base in the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine when the amplification product is detected; A method comprising:

[34] A method for determining the susceptibility of mycobacteria to macrolide antibiotics, comprising the method described in

[33] , determining that the acid-fast bacterium is susceptible to a macrolide antibiotic when the amplification product is detected; A method comprising:

[35] 1. A method for determining that the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in an acid-fast bacterium, in an erm(41) gene, is cytosine, comprising: Incubating the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined with the probe according to any one of

[29] to

[32] under conditions allowing hybridization; detecting hybridization of the genomic DNA or the polynucleotide with the probe; and determining that the base in the erm(41) gene in the genomic DNA of the acid-fast bacterium to be determined is cytosine when the hybridization is detected; A method comprising:

[36] A method for determining the susceptibility of mycobacteria to macrolide antibiotics, comprising the method described in

[35] , determining that the acid-fast bacterium is susceptible to a macrolide antibiotic when the hybridization is detected; A method comprising:

[0014]

[37] Use of a primer set according to any one of [5] to [8], a kit according to

[10] , or a probe according to any one of

[11] to

[14] for detecting an indicator base sequence in acid-fast bacteria that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene is cytosine, but is not present when the base is thymine.

[38] Use of a primer set according to any one of [5] to [8], a kit according to

[10] , or a probe according to any one of

[11] to

[14] to determine whether the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 in the erm(41) gene in acid-fast bacteria is cytosine or thymine.

[39] Use of a primer set according to any one of [5] to [8], a kit according to

[10] , or a probe according to any one of

[11] to

[14] for determining the susceptibility of mycobacteria to macrolide antibiotics.

[40] Use of a primer set according to any one of [5] to [8], a kit according to

[10] , or a probe according to any one of

[11] to

[14] to determine that the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 of the erm(41) gene in acid-fast bacteria is cytosine.

[0015] This specification includes the disclosure of Japanese Patent Application No. 2020-066277, from which this application claims priority. [Effects of the Invention]

[0016] According to one or more embodiments of the present invention, it is possible to easily determine whether the base at position 28 of the erm(41) gene in an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine. Furthermore, according to one or more embodiments of the present invention, it is possible to easily determine that the base at position 28 of the erm(41) gene in an acid-fast bacterium is cytosine. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows a schematic diagram of a nucleic acid detection device 10. 1: solid phase carrier, 2: conjugate pad (labeled substance holding portion), 3: sample pad (sample receiving portion), 4: absorption pad, 5: substrate, 6: portion including tag capture means. [Figure 2] Figure 2 shows the results of Example 2, in which the genomic DNA of different mycobacterial strains was used as a template and the PCR amplification products were detected by nucleic acid chromatography using a primer set that can specifically amplify the indicator base sequence shown in Sequence No. 2. [Figure 3] 3 shows the results of agarose gel electrophoresis of PCR amplification products obtained in Example 2 using the genomic DNA of Mycobacteroides abscessus ATCC19977 (T28 strain) and Mycobacteroides abscessus LRC18036 (C28 strain) as templates and primer sets 2 to 7 capable of specifically amplifying different regions of the index nucleotide sequence shown in SEQ ID NO: 2. In FIG. 3, A shows the results of agarose gel electrophoresis of PCR amplification products obtained using primer set 2, primer set 3, primer set C, primer set 4, primer set D, primer set 5, primer set E, primer set 6, and primer set F. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below.

[0019] <Terminology> In the present invention, the term polynucleotide or nucleic acid refers to DNA or RNA, typically DNA. The term polynucleotide or nucleic acid is not particularly limited by the number of bases and also encompasses oligonucleotides. In the present invention, the polynucleotide or nucleic acid contained in a primer or probe is typically a polymer of natural nucleotides. A natural nucleotide is a nucleotide composed of natural bases such as adenine, thymine, guanine, cytosine, or uracil, a sugar moiety of deoxyribose or ribose, and a phosphate group, each of which is free from artificial modifications. Natural nucleotides are usually D-nucleotides. A D-nucleotide refers to a nucleotide whose sugar moiety is D-deoxyribose or ribose.

[0020] The statement that nucleotide sequence X is "hybridizable" with nucleotide sequence Y means that a polynucleotide (particularly a DNA fragment) containing nucleotide sequence X hybridizes to a polynucleotide (particularly a DNA fragment) containing nucleotide sequence Y under stringent conditions, but does not hybridize to a polynucleotide not having nucleotide sequence Y. In other words, "hybridize" refers to specific hybridization. Here, "stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed, and can be appropriately determined by referring to, for example, Green and Sambrook, Molecular Cloning, 4th Ed. (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set by the temperature and salt concentration in the solution during Southern hybridization, and the temperature and salt concentration in the solution during the washing step of Southern hybridization. More specifically, stringent conditions in the hybridization step include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 40 to 68°C, preferably 40 to 65°C. More specifically, hybridization can be carried out in 1 to 7×SSC (1×SSC is 150 mM sodium chloride, 15 mM monosodium citrate, pH 7.2), 0.02 to 3% SDS, and at a temperature of 40 to 60°C. A washing step may also be carried out after hybridization, and the washing step can be carried out, for example, in 0.1 to 2×SSC, 0.1 to 0.3% SDS, and at a temperature of 50 to 65°C.

[0021] Specific examples of stringent conditions include hybridization of a DNA fragment containing base sequence X with a DNA fragment containing base sequence Y overnight (approximately 8 to 16 hours) in a hybridization solution containing 5xSSC, 0.1% (w / v) N-lauroylsarcosine, and 0.02% (w / v) SDS, followed by washing twice for 15 minutes using a washing solution containing 0.1 to 0.5xSSC and 0.1% (w / v) SDS, preferably 0.1xSSC and 0.1% (w / v) SDS. The temperature for hybridization and washing is preferably 50°C or higher, more preferably 65°C or higher. The hybridization solution may further contain, preferably, 0.5 to 2% (w / v) of a blocking reagent for nucleic acid hybridization. If a DNA probe containing base sequence X hybridizes with a DNA fragment containing base sequence Y after hybridization and washing under these conditions, it can be said that base sequence X is "hybridizable" with base sequence Y.

[0022] When base sequence X is hybridizable to base sequence Y, the polynucleotide (particularly DNA) containing base sequence X and the polynucleotide (particularly DNA) containing base sequence Y need only be a combination that can form sufficient hydrogen bonds to hybridize and form a stable double strand under the annealing conditions of a nucleic acid amplification reaction, and do not need to be completely complementary to each other. For example, there may be several mismatches between base sequence X and base sequence Y, such as one mismatch every 10 bases, one mismatch every 20 bases, or one mismatch every 30 bases.

[0023] When base sequence X is hybridizable to base sequence Y, or when expressed as "base sequence Y hybridizable to base sequence X", it preferably satisfies one or more of the following relationships (A) to (C). (A) The complementary base sequence of base sequence X is identical to base sequence Y. Note that if one of the complementary base sequence of base sequence X and base sequence Y is a DNA base sequence and the other is an RNA base sequence, thymine in one and uracil in the other are considered to be the same base. (B) The base sequence Y is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the complementary base sequence of the base sequence X. (C) The base sequence Y has an identity of 80% or more with the complementary base sequence of the base sequence X.

[0024] In the above (B), "one or several" preferably refers to one to five, more preferably one to four, more preferably one to three, particularly preferably one or two, and most preferably one.

[0025] In (C), the identity value indicates a value calculated using software (e.g., FASTA, DANASYS, and BLAST) that calculates the identity between multiple base sequences under default settings. The identity value of base sequences is calculated by aligning a pair of base sequences to maximize the degree of identity, calculating the number of matching bases, and calculating the ratio of the number of matching bases to the total number of bases in the compared base sequences. Here, if there are gaps, the total number of bases mentioned above is the number of bases counted with one gap as one base. For details on how to determine identity, see, for example, Altschul et al., Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990.

[0026] In (C) above, the identity is more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more. Of the above (A) to (C), the above (A) is particularly preferred.

[0027] In the present invention, the method for producing the polynucleotide contained in the primer and / or probe is not particularly limited, and the polynucleotide may be produced using a polynucleotide synthesizer or a custom synthesis service.

[0028] <Nucleic acid amplification reaction> When a nucleic acid amplification reaction is carried out using a primer set according to one or more embodiments of the present invention, the nucleic acid amplification reaction may be carried out using a thermostable polymerase or a strand displacement polymerase. The polymerase refers to a nucleic acid polymerase, and is a DNA polymerase or an RNA polymerase, preferably a DNA polymerase.

[0029] Examples of nucleic acid amplification reactions using a thermostable polymerase include polymerase chain reaction (PCR). Commercially available DNA polymerases can be used as the thermostable polymerase, and TaKaRa Ex Taq (registered trademark) and the like can be suitably used. The temperature, time, buffer composition, and the like can be appropriately selected depending on the DNA polymerase used and the concentration of each primer. The PCR conditions for the denaturation, annealing, and extension steps, such as time, temperature, buffer composition, substrate nucleotide concentration, and number of cycles, can be appropriately set taking into account factors such as the selected DNA polymerase, primer sequence, number of bases in the target nucleic acid, and template concentration.

[0030] Strand displacement polymerases are enzymes that synthesize new DNA strands while dissociating hydrogen bonds in double-stranded nucleic acids, including target nucleic acids, and examples include φ29 DNA polymerase, Bst DNA polymerase, Klenow fragment of DNA polymerase I, Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, 96-7 DNA polymerase, Aac DNA polymerase, and Csa DNA polymerase. Strand displacement polymerases do not require dissociation of the double strand, allowing isothermal nucleic acid amplification.

[0031] As a nucleic acid amplification reaction using a strand displacement polymerase, an isothermal nucleic acid amplification method is preferred, and examples thereof include the RPA method (Recombinase Polymerase Amplification), the TRIAmp method (Tandem Repeat-mediated Isothermal Amplification), the LAMP method (Loop-mediated isothermal amplification), and the HDA method (Helicase-dependent amplification).

[0032] Isothermal nucleic acid amplification using a strand displacement polymerase proceeds by incubating a template nucleic acid, a primer, a strand displacement polymerase, and substrate nucleotides in the presence of each other at a temperature at which the amplification primer can form stable base pairs with the template nucleic acid and at which the enzyme activity can be exerted. The conditions for the nucleic acid amplification reaction using the isothermal nucleic acid amplification method, such as the temperature, buffer composition, substrate nucleotide concentration, and reaction time, can be appropriately set taking into account factors such as the selected strand displacement polymerase, primer sequence, the number of bases in the target nucleic acid, and template nucleic acid concentration.

[0033] The term "target nucleic acid" refers to a nucleic acid containing a base sequence among index base sequences to be detected and / or amplified, or a nucleic acid containing a complementary base sequence of the base sequence to be detected and / or amplified. The target nucleic acid may exist as a double-stranded nucleic acid together with its complementary strand. One strand of a double-stranded target nucleic acid may also be referred to as the "target nucleic acid." Either the nucleic acid to be detected or its complementary strand may be referred to as the "target nucleic acid." In other words, in the present invention, "detecting a target nucleic acid" or "amplifying a target nucleic acid" encompasses both detecting or amplifying a target nucleic acid with the aim of detecting or amplifying the target nucleic acid itself, or detecting or amplifying the target nucleic acid, the complementary strand of the target nucleic acid, or a double-stranded nucleic acid consisting of the target nucleic acid and its complementary strand, with the aim of detecting or amplifying the complementary strand of the target nucleic acid or the double-stranded nucleic acid consisting of the target nucleic acid and its complementary strand.

[0034] When performing a nucleic acid amplification reaction, the nucleic acid serving as a template may be DNA or RNA as long as it partially contains the target base sequence and / or its complementary base sequence, but preferably it is DNA. Usually, the nucleic acid serving as a template for the nucleic acid amplification reaction is double-stranded DNA consisting of a polynucleotide chain containing at least a part of the target nucleic acid and the complementary strand of the polynucleotide chain.

[0035] The nucleic acid serving as a template may be natural or artificially synthesized. For example, it may be a natural nucleic acid extracted from a biological sample, or it may be one amplified by a nucleic acid amplification reaction such as PCR, or cDNA synthesized by a reverse transcription reaction, etc.

[0036] <Mycobacterium> In this specification, the "Mycobacterium" to be discriminated means acid-fast, non-motile Gram-positive bacilli. In this specification, "Mycobacterium" may include "Nontuberculous mycobacteria (NTM)", especially "Mycobacterium belonging to the Mycobacteroides abscessus complex". As described above, the Mycobacteroides abscessus complex includes 3 subspecies. One or more embodiments of the present invention are preferably used to detect the mutation of the 28th base of the erm(41) gene in the Mycobacteroides abscessus complex, and it is particularly preferably used to detect the mutation of the 28th base of the erm(41) gene in Mycobacteroides abscessus subspecies abscessus and Mycobacteroides abscessus subspecies bolletii. Note that the genus Mycobacteroides may sometimes be described as the genus Mycobacterium.

[0037] <erm(41) gene> The erm(41) gene encodes erythromycin ribosomal methyltransferase. The erm(41) gene expresses erythromycin ribosomal methyltransferase in the presence of macrolide antibiotics such as clarithromycin, thereby inducing resistance to macrolide antibiotics.

[0038] The erm(41) gene contained in the genomic DNA of the type strain of Mycobacteroides abscessus complex has the nucleotide sequence of SEQ ID NO: 1. In the nucleotide sequence of SEQ ID NO: 1, the 28th base is thymine. Mycobacteria whose erm(41) gene has the nucleotide sequence of SEQ ID NO: 1 are resistant bacteria that can induce resistance to macrolide antibiotics.

[0039] On the other hand, approximately 10% of Mycobacteroides abscessus complex strains have a mutation in the erm(41) gene at the 28th base in the nucleotide sequence of SEQ ID NO: 1, from thymine to cytosine. Mycobacteria with this genotype cannot induce resistance to macrolide antibiotics and are susceptible to these antibiotics.

[0040] Some acid-fast bacteria belonging to the Mycobacteroides abscessus complex are strains partially deleted in the erm(41) gene, i.e., they lack a portion of the full-length nucleotide sequence of the erm(41) gene set forth in SEQ ID NO: 1. Even strains partially deleted in the erm(41) gene still contain the nucleotide sequence upstream of the 28th base in the nucleotide sequence set forth in SEQ ID NO: 1, and some strains contain this nucleotide as thymine and some as cytosine.

[0041] In the following description, a type of erm(41) gene in which the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 is thymine will be referred to as the "normal type," "erm(41)T28," or "T28." An acid-fast bacterium strain in which the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 is thymine will be referred to as the "T28 strain." The base sequence of the erm(41)T28 gene refers to one in which the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 is thymine. The base sequence at other positions may be identical to the base sequence of SEQ ID NO: 1, or may be one in which one or more bases in the base sequence of SEQ ID NO: 1 have been deleted, substituted, added, and / or inserted, or a partial sequence downstream of position 28 in the erm(41) gene may be deleted, as described above.

[0042] In the following description, the form of the erm(41) gene in which the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 is cytosine will be referred to as a "mutant," "erm(41)C28," or "C28." An acid-fast bacterial strain in which the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 is cytosine will be referred to as a "C28 strain." The base sequence of the erm(41)C28 gene refers to one in which the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 is cytosine. The base sequence at other positions may be identical to the base sequence of SEQ ID NO: 1, or may be one in which one or more bases in the base sequence of SEQ ID NO: 1 have been deleted, substituted, added, and / or inserted, or a partial sequence downstream of position 28 in the erm(41) gene may be deleted, as described above.

[0043] In the above two paragraphs, "one or several" preferably refers to one to five, more preferably one to four, more preferably one to three, particularly preferably one or two, and most preferably one.

[0044] <Method 1 for determining the erm(41) genotype of mycobacteria> A first embodiment of the present invention comprises: 1. A method for determining whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine, comprising: An indicator base sequence is detected in the genomic DNA of the acid-fast bacterium to be evaluated, the indicator base sequence being present in a region of the genomic DNA other than the erm(41) gene when the base in the erm(41) gene is cytosine, and not present when the base is thymine. Including, detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine; The absence of detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is thymine. Regarding the method.

[0045] According to the method of the first embodiment, it is possible to determine whether an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is a C28 strain or a T28 strain by detecting an indicator base sequence present in a region of the genomic DNA other than the erm(41) gene, which is specific to the case where the acid-fast bacterium is a C28 strain, rather than by detecting a single-base mutation in the erm(41) gene. The method of the first embodiment does not require direct detection of a single-base mutation, and can be easily carried out because it is possible to determine whether the strain is a C28 strain or a T28 strain by simply detecting the presence or absence of an indicator base sequence in the genomic DNA.

[0046] The method according to the first embodiment can also be used to determine whether mycobacteria, particularly mycobacteria belonging to the Mycobacteroides abscessus complex, are susceptible to macrolide antibiotics. The method for determining the susceptibility of mycobacteria to macrolide antibiotics comprises detecting the indicator base sequence in the genomic DNA of the mycobacterium to be determined, where detection of the indicator base sequence indicates that the mycobacterium is susceptible to macrolide antibiotics, and absence of detection of the indicator base sequence indicates that the mycobacterium is not susceptible to macrolide antibiotics.

[0047] The indicator sequence can also be used as an indicator to determine that the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 of the erm(41) gene in acid-fast bacteria is cytosine.

[0048] The present inventors have surprisingly found that a region having the nucleotide sequence of SEQ ID NO: 2 is present specifically in the genomic DNA of the C28 strain, but is not present in the genomic DNA of the T28 strain.

[0049] The nucleotide sequence of SEQ ID NO: 2 is a nucleotide sequence in which, from the 5' end to the 3' end, the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence of SEQ ID NO: 6, the nucleotide sequence of SEQ ID NO: 7, the nucleotide sequence of SEQ ID NO: 8, the nucleotide sequence of SEQ ID NO: 9, the nucleotide sequence of SEQ ID NO: 10, and the nucleotide sequence of SEQ ID NO: 11 are linked in this order. The 5'-end and 3'-end bases of each of the nucleotide sequences of SEQ ID NOs: 3 to 11 are shown in the table below relative to the nucleotide sequence of SEQ ID NO: 2.

[0050] [Table 1]

[0051] Of the base sequence of SEQ ID NO: 2, the base sequence of SEQ ID NO: 3, the base sequence of SEQ ID NO: 5, the base sequence of SEQ ID NO: 7, the base sequence of SEQ ID NO: 9, and the base sequence of SEQ ID NO: 11 are particularly preferred as indicators for determining the C28 strain, because they are base sequences that do not have relatively highly homologous base sequences in the genomic DNA of the T28 strain.

[0052] The indicator base sequence may be any base sequence that is specifically present in the genomic DNA of the acid-fast bacterium C28 strain, and specifically, is preferably a first base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 2, or a second base sequence complementary to the first base sequence. The first base sequence is more preferably a base sequence of 20 or more consecutive bases, 50 or more bases, 100 or more bases, 500 or more bases, or 1000 or more bases contained in the base sequence of SEQ ID NO: 2. The upper limit of the number of bases in the first base sequence is not particularly limited, but may be, for example, 3000 or less bases or 1500 or less bases.

[0053] More preferably, the first base sequence contains at least a portion of a base sequence of 10 or more consecutive bases, preferably 20 or more, 50 or more, 100 or more, 500 or more, or 1000 or more bases, contained in the base sequence of SEQ ID NO: 3, 5, 7, 9, or 11. Such a first base sequence or its complementary second base sequence is a base sequence to which no relatively highly homologous base sequence exists in the genomic DNA of the T28 strain, and therefore, the presence of such a base sequence enables highly accurate identification of the C28 strain.

[0054] In the method according to the first embodiment, the method for detecting the index base sequence is not particularly limited, and can be carried out, for example, by a method using a probe or primer as described below.

[0055] In the method according to the first embodiment, when the target base sequence is detected in the genomic DNA of the mycobacterium belonging to the Mycobacterium abscessus complex, it can be determined that the mycobacterium is strain C28, and when the target base sequence is not detected in the genomic DNA of the mycobacterium, it can be determined that the mycobacterium is strain T28.

[0056] <Probe for detecting a target base sequence specific to strain C28> The second embodiment of the present invention is In a mycobacterium belonging to the Mycobacterium abscessus complex, a probe for detecting a target base sequence that is present in a region other than the erm(41) gene of the genomic DNA when the base corresponding to the 28th position of the base sequence of SEQ ID NO: 1 of the erm(41) gene is cytosine and is not present when the base is thymine, A probe comprising a polynucleotide comprising a base sequence capable of hybridizing with a partial base sequence p1 of 10 or more consecutive bases included in the target base sequence relates to.

[0057] The target base sequence in the second embodiment can be selected from the same range as the target base sequence with respect to the first embodiment. The partial base sequence p1 is a region of the target base sequence that hybridizes with the probe according to the second embodiment.

[0058] The partial base sequence p1 is a continuous partial base sequence in the target base sequence, and may have any number of bases necessary to maintain specificity, and the number of bases may be 10 or more, more preferably 15 or more, more preferably 17 or more, more preferably 20 or more. The upper limit of the number of bases of the partial base sequence p1 is not particularly limited, but can be, for example, a partial base sequence of the target base sequence of 400 bases or less, 300 bases or less, 200 bases or less, 100 bases or less, or 50 bases or less.

[0059] More preferably, the partial base sequence p1 contains at least a portion of a base sequence of 10 or more consecutive bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 20 or more bases, for example, 400 or less bases, 300 or less bases, 200 or less bases, 100 or less bases, or 50 or less bases, contained in the base sequence of SEQ ID NO: 3, 5, 7, 9, or 11 or its complementary base sequence.

[0060] The partial base sequence p1 is Preferably (p1-12-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-18-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-20-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-22-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-24-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-26-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-28-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-13-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-19-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-21-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-23-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-25-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-27-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2 or its complementary base sequence, or (p1-29-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 or its complementary base sequence. and More preferably (p1-12-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 4629 to 4663 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-18-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 2361 to 2392 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-20-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 2629 to 2658 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-22-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 4049 to 4078 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-24-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 6540 to 6570 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-26-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 7483 to 7515 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-28-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 7634 to 7668 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-13-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 4699 to 4735 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-19-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 2677 to 2706 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-21-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 3544 to 3578 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-23-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 5066 to 5098 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-25-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 6571 to 6600 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-27-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 7936 to 7971 of the base sequence of SEQ ID NO: 2 or its complementary base sequence, or (p1-29-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, contained in the base sequence ranging from positions 8307 to 8341 of the base sequence of SEQ ID NO: 2 or its complementary base sequence. is.

[0061] The probe according to the second embodiment of the present invention can hybridize under stringent conditions to the region of the partial base sequence p1 in a polynucleotide containing the index base sequence. Therefore, the probe according to the second embodiment of the present invention can be used in nucleic acid hybridization methods (e.g., Southern hybridization) and real-time PCR methods (e.g., TaqMan™ method, Molecular Beacon method), etc.

[0062] The polynucleotide constituting the probe according to the second embodiment contains a base sequence identical or homologous to the complementary base sequence of the partial base sequence p1. Here, "homologous" means that the polynucleotide satisfies the above-mentioned condition (B) or (C), specifically, the polynucleotide satisfies the following condition (B1) or (C1): (B1) The base sequence of the polynucleotide constituting the probe according to the second embodiment is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the complementary base sequence of the partial base sequence p1. (C1) The base sequence of the polynucleotide constituting the probe according to the second embodiment has an identity of 80% or more with the complementary base sequence of the partial base sequence p1. The preferred ranges of "one or several" in (B1) and "identity" in (C1) are as described above in relation to (B) and (C).

[0063] The polynucleotide constituting the probe according to the second embodiment does not necessarily consist solely of a base sequence identical or homologous to the complementary base sequence of the partial base sequence p1 (hereinafter referred to as "base sequence q1"), and other base sequences may be added to either or both of the 5'-end and 3'-end of base sequence q1. It is particularly preferred that base sequence q1 is identical to the complementary base sequence of the partial base sequence p1.

[0064] The total number of bases in the polynucleotide constituting the probe according to the second embodiment is not particularly limited, and may be 8 or more, 10 or more, more preferably 15 or more, more preferably 20 or more, and preferably 400 or less, 300 or less, or 200 or less. When the probe is used in real-time PCR, the polynucleotide preferably has 50 or less bases, more preferably 40 or less, or 35 or less bases.

[0065] Specific examples of the polynucleotide constituting the probe according to the second embodiment include: A polynucleotide comprising a base sequence of 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, particularly preferably 20 or more consecutive bases, contained in any of the base sequences of SEQ ID NOs: 12, 13, 18 to 29, or their complementary base sequences. Examples include:

[0066] The probe according to the second embodiment may further comprise a labeling moiety, which is a tag or labeling substance capable of binding to a labeling substance, attached to the polynucleotide. Specific examples of such labeling moieties are described below with respect to primers. A complex formed by hybridization of a probe containing a labeling moiety with a polynucleotide containing the index base sequence can be easily detected using the labeling moiety as an index.

[0067] The probe according to the second embodiment may further comprise a binding moiety, which is a tag capable of binding to a solid support, attached to the polynucleotide. Specific examples of such binding moieties are described below for primers. A complex formed by hybridization of the probe containing the binding moiety with a polynucleotide containing the index base sequence can be immobilized on a solid support via the binding moiety, facilitating detection and isolation of the complex.

[0068] The probe containing the binding moiety may be provided in a state in which it has been immobilized on a solid support via the binding moiety in advance. The probe immobilized on the solid support can capture a polynucleotide containing the index base sequence.

[0069] When the probe according to the second embodiment is used in real-time PCR, it is preferable to label it with a labeling substance commonly used in real-time detection of amplification products. For example, a reporter fluorescent substance is linked to the 5' end of the polynucleotide used as the probe, and a quencher dye is linked to the 3' end. Examples of reporter fluorescent substances include carboxyfluorescein (FAM), hexachlorofluorescein (HEX), and tetrachlorofluorescein (TET). Examples of quencher dyes include fluorescent substances such as carboxytetramethylrhodamine (TAMRA), and non-fluorescent substances such as Black Hole Quencher dye (BHQ) and 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL).

[0070] The probe according to the second embodiment can be used in a method for determining whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine.

[0071] Specifically, this method involves: incubating the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined with the probe under conditions that allow hybridization; detecting hybridization of the genomic DNA or the polynucleotide with the probe; and determining that the base in the erm(41) gene in the genomic DNA of the acid-fast bacterium to be determined is cytosine when the hybridization is detected, and determining that the base is thymine when the hybridization is not detected; The method can include:

[0072] The probe according to the second embodiment can also be used in a method for determining the susceptibility of acid-fast bacteria, particularly acid-fast bacteria belonging to the Mycobacteroides abscessus complex, to macrolide antibiotics. incubating the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined with the probe under conditions that allow hybridization; detecting hybridization of the genomic DNA or the polynucleotide with the probe; and The method may include determining that the mycobacterium is susceptible to macrolide antibiotics if the hybridization is detected, and determining that the mycobacterium is not susceptible to macrolide antibiotics if the hybridization is not detected.

[0073] The probe according to the second embodiment can also be used in a method for determining that, in mycobacteria, the base corresponding to the 28th position of the nucleotide sequence of SEQ ID NO: 1 of the erm(41) gene is cytosine. Specifically, this method involves incubating genomic DNA of the mycobacteria to be determined or a polynucleotide derived from the genomic DNA of the mycobacteria to be determined and the probe under conditions allowing hybridization, detecting the hybridization between the genomic DNA or the polynucleotide and the probe, and when the hybridization is detected, determining that the base of the erm(41) gene in the genomic DNA of the mycobacteria to be determined is cytosine, which can be a method including the above steps.

[0074] Here, examples of the "polynucleotide derived from the genomic DNA of the mycobacteria to be determined" include a partial polynucleotide of a part of the genomic DNA, and an amplification product obtained by amplifying the whole genomic DNA or a part containing the target base sequence by nucleic acid amplification reaction.

[0075] In each of the above aspects, the "conditions allowing hybridization" are preferably the same as the conditions described as "stringent conditions".

[0076] <Primer set for detecting an index base sequence specific to strain C28> The third embodiment of the present invention is a primer set for detecting an index base sequence that exists in a region other than the erm(41) gene of genomic DNA when the base corresponding to the 28th position of the nucleotide sequence of SEQ ID NO: 1 of the erm(41) gene is cytosine and does not exist when it is thymine in mycobacteria belonging to the Mycobacterium abscessus complex, a first primer containing a polynucleotide having, at its 3' end, a base sequence f12 capable of hybridizing with the complementary base sequence of a partial base sequence f11 of 10 or more consecutive bases included in the index base sequence, a second primer containing a polynucleotide containing, at its 3' end, a base sequence r12 that is contained in the index base sequence and is located closer to the 3' end than the 3' end of the partial base sequence f11 and that is capable of hybridizing to a partial base sequence r11 of 10 or more consecutive bases; A primer set including Regarding.

[0077] The indicator base sequence may be any base sequence that is specifically present in the genomic DNA of the acid-fast bacterium C28 strain, and specifically, a third base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 2 is preferred. The third base sequence is more preferably a base sequence of 40 or more consecutive bases, 50 or more bases, 100 or more bases, 500 or more bases, or 1000 or more bases contained in the base sequence of SEQ ID NO: 2. The upper limit of the number of bases in the third base sequence is not particularly limited, but may be, for example, 3000 or less bases or 1500 or less bases.

[0078] The nucleotide sequence contained in the third nucleotide sequence, extending from the 5'-terminal nucleotide of the partial nucleotide sequence f11 to the 3'-terminal nucleotide of the partial nucleotide sequence r11, is hereinafter referred to as the "target sequence." The target sequence and its complementary nucleotide sequence are amplified by a nucleic acid amplification reaction using the primer set according to this embodiment. The target sequence preferably contains at least a portion of a nucleotide sequence of 20 or more consecutive nucleotides, preferably 40 or more, 50 or more, 100 or more, 500 or more, or 1000 or more nucleotides contained in the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, or 11. This target sequence is a nucleotide sequence to which no nucleotide sequence with a relatively high homology exists in the genomic DNA of the T28 strain, and therefore, the presence of the target sequence allows for highly accurate identification of the C28 strain.

[0079] The length of the target sequence is not particularly limited. To improve detection accuracy, the target sequence preferably contains a continuous portion of the index base sequence of at least 20 bases, more preferably at least 40 bases, at least 50 bases, at least 100 bases, at least 500 bases, or at least 1000 bases. The upper limit of the length of the target sequence is not particularly limited, but is usually 3000 bases or less or 1500 bases or less.

[0080] The partial base sequence f11 is located at the 5' end of the target sequence, and the partial base sequence r11 is located at the 3' end of the target sequence.

[0081] The length of the partial base sequence f11 and the partial base sequence r11 can each be 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably 20 bases or more, and can typically be 40 bases or less, 30 bases or less, or 27 bases or less.

[0082] The partial base sequence f11 is Preferably, (f11-12-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2; (f11-18-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2; (f11-20-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2; (f11-22-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2; (f11-24-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2; (f11-26-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2, or (f11-28-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 and More preferably, (f11-12-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4629 to 4661 of the base sequence of SEQ ID NO: 2; (f11-18-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2361 to 2390 of the base sequence of SEQ ID NO: 2; (f11-20-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2629 to 2656 of the base sequence of SEQ ID NO: 2; (f11-22-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4049 to 4076 of the base sequence of SEQ ID NO: 2; (f11-24-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 6540 to 6568 of the base sequence of SEQ ID NO: 2; (f11-26-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 7483 to 7513 of the base sequence of SEQ ID NO: 2, or (f11-28-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 7634 to 7666 of the base sequence of SEQ ID NO: 2 and Particularly preferably, (f11-12-3) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4629 to 4658 of the base sequence of SEQ ID NO: 2; (f11-18-3) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2361 to 2387 of the base sequence of SEQ ID NO: 2; (f11-20-3) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2629 to 2653 of the base sequence of SEQ ID NO: 2; (f11-22-3) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4049 to 4073 of the base sequence of SEQ ID NO: 2; (f11-24-3) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 6540 to 6565 of the base sequence of SEQ ID NO: 2; (f11-26-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 7483 to 7510 of the base sequence of SEQ ID NO: 2, or (f11-28-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 7634 to 7663 of the base sequence of SEQ ID NO: 2 is.

[0083] In the above (f11-12-1) to (f11-28-1), the "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases" is preferably a "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases extending from the 1st to 12th bases from the 3'-end toward the 5'-end."

[0084] In the above (f11-12-2) to (f11-28-2), the "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases" is preferably a "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases extending from the first to fifth bases from the 3'-end toward the 5'-end."

[0085] In the above (f11-12-3) to (f11-28-3), the "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases" is preferably "a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases extending from the first or second base from the 3'-end toward the 5'-end." Here, the "first or second base from the 3'-end" is more preferably the "base at the 3'-end."

[0086] The partial base sequence r11 is Preferably, (r11-13-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2; (r11-19-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2; (r11-21-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2; (r11-23-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2; (r11-25-1) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2; (r11-27-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2, or (r11-29-1) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 and More preferably, (r11-13-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4701 to 4735 of the base sequence of SEQ ID NO: 2; (r11-19-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2679 to 2706 of the base sequence of SEQ ID NO: 2; (r11-21-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 3546 to 3578 of the base sequence of SEQ ID NO: 2; (r11-23-2) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 5068 to 5098 of the base sequence of SEQ ID NO: 2; (r11-25-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, included in the base sequence ranging from positions 6573 to 6600 of the base sequence of SEQ ID NO: 2; (r11-27-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 7938 to 7971 of the base sequence of SEQ ID NO: 2, or (r11-29-2) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 8309 to 8341 of the base sequence of SEQ ID NO: 2 and Particularly preferably, (r11-13-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 4704 to 4735 of the base sequence of SEQ ID NO: 2; (r11-19-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 2682 to 2706 of the base sequence of SEQ ID NO: 2; (r11-21-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 3549 to 3578 of the base sequence of SEQ ID NO: 2; (r11-23-3) a base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 5071 to 5098 of the base sequence of SEQ ID NO: 2; (r11-25-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 6576 to 6600 of the base sequence of SEQ ID NO: 2; (r11-27-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 7941 to 7971 of the base sequence of SEQ ID NO: 2, or (r11-29-3) A base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases included in the base sequence ranging from positions 8312 to 8341 of the base sequence of SEQ ID NO: 2 is.

[0087] In the above (r11-13-1) to (r11-29-1), the "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases" is preferably a "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases extending from the 1st to 12th bases from the 5'-end toward the 3'-end."

[0088] In the above (r11-13-2) to (r11-29-2), the "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases" is preferably a "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases extending from the first to fifth bases from the 5'-end toward the 3'-end."

[0089] In the above (r11-13-3) to (r11-29-3), the "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases" is preferably a "base sequence of 10 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases from the first or second base from the 5' end toward the 3' end." The "first or second base from the 5' end" is more preferably the "5' end base."

[0090] The partial base sequence f11 and the partial base sequence r11 may be a combination in which the base at the 3' end of the partial base sequence f11 is located closer to the 5' end (upstream) of the base at the 5' end of the partial base sequence r11 in the base sequence of SEQ ID NO: 2. Preferably, when the partial base sequence f11 is (f11-12-1), (f11-12-2), or (f11-12-3), the partial base sequence r11 is (r11-13-1), (r11-13-2), (r11-13-3), (r11-23-1), (r11-23-2), or (r11-23-3). In this case, the partial base sequence of SEQ ID NO: 7 can be amplified. Preferably, when the partial base sequence f11 is (f11-18-1), (f11-18-2), or (f11-18-3), the partial base sequence r11 is (r11-19-1), (r11-19-2), (r11-19-3), (r11-21-1), (r11-21-2), or (r11-21-3). In this case, the partial base sequence of SEQ ID NO: 5 can be amplified. Preferably, when the partial base sequence f11 is (f11-20-1), (f11-20-2), or (f11-20-3), the partial base sequence r11 is (r11-19-1), (r11-19-2), (r11-19-3), (r11-21-1), (r11-21-2), or (r11-21-3). In this case, the partial base sequence of SEQ ID NO: 5 can be amplified. Preferably, when the partial base sequence f11 is (f11-22-1), (f11-22-2), or (f11-22-3), the partial base sequence r11 is (r11-13-1), (r11-13-2), (r11-13-3), (r11-23-1), (r11-23-2), or (r11-23-3). In this case, the partial base sequence of SEQ ID NO: 7 can be amplified. Preferably, when the partial base sequence f11 is (f11-24-1), (f11-24-2), or (f11-24-3), the partial base sequence r11 is (r11-25-1), (r11-25-2), or (r11-25-3). In this case, the partial base sequence of SEQ ID NO: 9 can be amplified. Preferably, when the partial base sequence f11 is (f11-26-1), (f11-26-2), or (f11-26-3), the partial base sequence r11 is (r11-27-1), (r11-27-2), (r11-27-3), (r11-29-1), (r11-29-2), or (r11-29-3). In this case, the partial base sequence of SEQ ID NO: 11 can be amplified. Preferably, when the partial base sequence f11 is (f11-28-1), (f11-28-2), or (f11-28-3), the partial base sequence r11 is (r11-27-1), (r11-27-2), (r11-27-3), (r11-29-1), (r11-29-2), or (r11-29-3). In this case, the partial base sequence of SEQ ID NO: 11 can be amplified.

[0091] The polynucleotide in the first primer can be designed to contain, at its 3'-end, a base sequence f12 that can hybridize to the complementary base sequence of the partial base sequence f11. The polynucleotide in the first primer may contain base sequence f12 at its 3'-end, and may further contain another base sequence attached to the 5'-end of base sequence f12. The total length of the polynucleotide in the first primer is not particularly limited, but may be, for example, 10 bases or more, more preferably 17 bases or more, more preferably 20 bases or more, and typically 40 bases or less, 30 bases or less, or 27 bases or less.

[0092] When the base sequence f12 can hybridize to a complementary base sequence of the partial base sequence f11, the partial base sequence f11 and the base sequence f12 are identical or homologous. Here, "homologous" means that the condition (B) or (C) above is satisfied, and specifically, the following condition (B2) or (C2) is satisfied. (B2) The base sequence f12 is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the partial base sequence f11. (C2) The base sequence f12 has an identity of 80% or more with the partial base sequence f11.

[0093] The preferred ranges of "one or several" in (B2) and "identity" in (C2) are as described above in relation to (B) and (C).

[0094] The polynucleotide in the second primer can be designed to contain a base sequence r12 at its 3' end that is hybridizable with the partial base sequence r11. The polynucleotide in the second primer may contain the base sequence r12 at its 3' end, and another base sequence may be added to the 5' end of the base sequence r12. The total length of the polynucleotide in the second primer is not particularly limited, but can be, for example, 10 bases or more, more preferably 17 bases or more, more preferably 20 bases or more, and typically 40 bases or less, 30 bases or less, or 27 bases or less.

[0095] When the base sequence r12 can hybridize with the partial base sequence r11, the complementary base sequence of the partial base sequence r11 and the base sequence r12 are identical or homologous. Here, "homologous" means that the condition (B) or (C) above is satisfied, and specifically, the following condition (B3) or (C3) is satisfied. (B3) The base sequence r12 is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the complementary base sequence of the partial base sequence r11. (C3) The base sequence r12 has an identity of 80% or more with the complementary base sequence of the partial base sequence r11.

[0096] The preferred ranges of "one or several" in (B3) and "identity" in (C3) are as described above in relation to (B) and (C).

[0097] The combination of the first primer and the second primer is not particularly limited, and can be such that the target region of the genomic DNA can be amplified as a polynucleotide fragment by a nucleic acid amplification reaction.

[0098] Preferable examples of the primer set according to the third embodiment include: The first primer is (C28f) A polynucleotide of 40 bases or less containing, at the 3' end, a base sequence C28fb of 10 or more consecutive bases contained in the base sequence C28fa identical or homologous to the base sequence shown in SEQ ID NO: 12, 18, 20, 22, 24, 26, or 28 a primer comprising The second primer is (C28r) A polynucleotide of 40 bases or less containing, at the 3' end, a base sequence C28rb of 10 or more consecutive bases contained in the base sequence C28ra identical or homologous to the base sequence shown in SEQ ID NO: 13, 19, 21, 23, 25, 27, or 29 is a primer comprising An example is a primer set.

[0099] In the (C28f), the base sequence C28fa is identical to or homologous to the base sequence of SEQ ID NO: 12, 18, 20, 22, 24, 26, or 28, i.e., satisfies the relationship (B) or (C) (the base sequence of SEQ ID NO: 12, 18, 20, 22, 24, 26, or 28 corresponds to base sequence X, and the base sequence C28fa corresponds to base sequence Y).

[0100] In the above (C28f), the base sequence C28fa is preferably a base sequence that is identical to the base sequence shown in SEQ ID NO: 12, 18, 20, 22, 24, 26, or 28 in a portion of preferably 3 or more consecutive bases, more preferably 5 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 12 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases from the 3' end, and the remainder is homologous (i.e., satisfies the relationship (B) or (C) above (the remainder of the base sequence of SEQ ID NO: 12, 18, 20, 22, 24, 26, or 28 corresponds to base sequence X, and the remainder of base sequence C28fa corresponds to base sequence Y)), and is preferably identical to the base sequence shown in SEQ ID NO: 12, 18, 20, 22, 24, 26, or 28.

[0101] The base sequence C28fb is preferably a base sequence of 12 or more, 15 or more, 17 or more, or 20 or more consecutive bases contained in the base sequence C28fa, and more preferably a consecutive base sequence starting from the 3'-terminal base of the base sequence C28fa.

[0102] The base sequence C28fb is more preferably identical to the base sequence C28fa.

[0103] In the (C28r), the base sequence C28ra is identical to or homologous to the base sequence of SEQ ID NO: 13, 19, 21, 23, 25, 27, or 29, i.e., satisfies the relationship (B) or (C) (the base sequence of SEQ ID NO: 13, 19, 21, 23, 25, 27, or 29 corresponds to base sequence X, and the base sequence C28ra corresponds to base sequence Y).

[0104] In the above (C28r), the base sequence C28ra is preferably a base sequence that is identical to the base sequence shown in SEQ ID NO: 13, 19, 21, 23, 25, 27, or 29 in a portion of preferably 3 or more consecutive bases, more preferably 5 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 12 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases from the 3' end, and the remainder is homologous (i.e., satisfies the relationship (B) or (C) above (the remainder of the base sequence of SEQ ID NO: 13, 19, 21, 23, 25, 27, or 29 corresponds to base sequence X, and the remainder of base sequence C28ra corresponds to base sequence Y)), and is preferably identical to the base sequence shown in SEQ ID NO: 13, 19, 21, 23, 25, 27, or 29.

[0105] The base sequence C28rb is preferably a base sequence of 12 or more, 15 or more, 17 or more, or 20 or more consecutive bases contained in the base sequence C28ra, and more preferably a consecutive base sequence starting from the 3'-terminal base of the base sequence C28ra.

[0106] The base sequence C28rb is more preferably identical to the base sequence C28ra.

[0107] The first primer may consist solely of the polynucleotide, or may further comprise a labeling moiety or a binding moiety, as described below. The polynucleotide and the labeling moiety or the binding moiety can be chemically linked via a suitable spacer, as described below. In the first primer, the position at which one of the labeling moiety and the binding moiety is linked to the polynucleotide is not particularly limited as long as it does not inhibit annealing of the polynucleotide to a polynucleotide containing the target region or its complementary strand and extension in a nucleic acid amplification reaction, but is preferably the 5' end of the polynucleotide.

[0108] The second primer may consist solely of the polynucleotide, or may further comprise a labeling moiety or a binding moiety, as described below. The polynucleotide and the labeling moiety or the binding moiety can be chemically linked via a suitable spacer, as described below. In the second primer, the position at which one of the labeling moiety and the binding moiety is linked to the polynucleotide is not particularly limited, as long as it does not inhibit annealing of the polynucleotide with a polynucleotide containing the target region or its complementary strand and extension in a nucleic acid amplification reaction, but is preferably the 5' end of the polynucleotide.

[0109] When at least one of the first primer and the second primer contains a label, a nucleic acid amplification reaction using this primer will yield an amplification product containing the label, making it easy to detect the amplification product.

[0110] When at least one of the first primer and the second primer contains a binding moiety, a nucleic acid amplification reaction using this primer will yield an amplification product containing the binding moiety, making it possible to immobilize the amplification product on a solid phase support and facilitating detection of the amplification product.

[0111] More preferably, one of the first primer and the second primer further comprises a labeling moiety, and the other further comprises a binding moiety. In a nucleic acid amplification reaction using the primer set of this embodiment, a double-stranded amplification product containing the labeling moiety and the binding moiety is obtained, which facilitates detection by nucleic acid chromatography.

[0112] A primer set including a labeling portion and a binding portion will be described below.

[0113] (Sign part) The labeling moiety is either a tag capable of binding to a labeling substance or a labeling substance, and is preferably a tag capable of binding to a labeling substance. In this specification, a tag capable of binding to a labeling substance may be referred to as a labeling tag. When the labeling moiety is a labeling tag, the tag contained at one end of the amplification product can be labeled by contacting the amplification product of the nucleic acid amplification reaction with the labeling substance. When the labeling moiety is a labeling substance, an amplification product containing the labeling substance at one end can be obtained as the amplification product of the nucleic acid amplification reaction.

[0114] The labeling substance may be any substance that allows the detection of the amplification product, but is preferably one that allows the amplification product to be visually detected. Examples of such labeling substances include colored particles, dyes, enzymes (peroxidase, alkaline phosphatase, luciferase, etc.), with colored particles being preferred. "Colored particles" include, but are not limited to, metal particles (e.g., gold, silver, copper, platinum, etc.), metal rods, colored latex particles, and silica nanoparticles containing dyes. The size of the labeling substance may be such that it does not interfere with the capture of the amplification product on the solid-phase carrier described below. The labeling substance preferably exhibits good color development during detection and can be appropriately selected so that its size is smaller than the pore size of the solid-phase carrier described below or various porous members of nucleic acid detection devices equipped with the solid-phase carrier. For example, the size of the labeling substance can be approximately 500 nm or less, preferably approximately 0.1 nm to 250 nm, and more preferably approximately 1 nm to 100 nm. Fluorescent dyes (fluorescein, cyanine, etc.) can also be used as dyes, but in this case it is preferable to detect them by irradiating them with light of the excitation wavelength of each fluorescent dye.

[0115] Labeling tags that can be used as labeling moieties are not particularly limited as long as they can bind to the labeling substance and can be selected appropriately depending on the structure of the labeling substance. Examples of suitable labeling tags include nucleic acids (DNA, RNA, etc.), proteins, peptides, and other compounds (e.g., low-molecular-weight compounds such as biotin, fluorescein isothiocyanate (FITC), and digoxigenin (DIG)), as well as combinations thereof. One preferred form of labeling tag is one that contains or consists of a polynucleotide. The polynucleotide that can be contained in the labeling tag is not particularly limited as long as it does not substantially interfere with the nucleic acid amplification reaction using the primer set. It is preferably a polynucleotide having, for example, 5 to 50 bases, preferably 10 to 35 bases. More preferably, a polynucleotide containing (or consisting of) the base sequence set forth in SEQ ID NO: 14 or 15, or a partial base sequence thereof, or a complementary base sequence thereof can be used. Another preferred form of labeling tag is one that consists of a low-molecular-weight compound such as biotin, FITC, or DIG.

[0116] When the labeling moiety is the labeling tag described above, the labeling substance and the labeling tag may be directly or indirectly bound. The binding method can be appropriately selected depending on the combination of the labeling substance and the labeling tag used. For example, when the labeling tag contains a polynucleotide, the labeling substance can be indirectly bound to the labeling tag by binding the labeling substance to a polynucleotide capable of hybridizing to the polynucleotide (e.g., a polynucleotide containing a sequence complementary to the base sequence of the polynucleotide) and then hybridizing the two polynucleotides. The labeling substance and the polynucleotide may be bound via a peptide, protein, nucleic acid, or an appropriate functional group. Hybridization conditions are not particularly limited as long as they allow hybridization to occur. For example, the hybridization can be carried out at 20°C to 40°C in a buffer solution containing 10 mM to 50 mM phosphate (pH 6 to 7). To enhance hybridization efficiency, the buffer solution may further contain a salt such as sodium chloride.

[0117] Furthermore, when the labeling tag is a low molecular weight compound, it can be labeled with a labeling substance linked to a binding substance such as a protein that specifically binds to it (for example, avidin that binds to biotin, or a protein that binds to FITC), an antibody (for example, an anti-DIG antibody), an aptamer, etc. In this case, the labeling tag and the binding substance can be bound using various near-neutral buffer solutions.

[0118] The labeling moiety (labeling tag or labeling substance) can be bound to the polynucleotide contained in the first primer or the polynucleotide contained in the second primer by any means, and can be bound directly or indirectly. However, when at least the portion of the labeling moiety that is connected to the polynucleotide consists of a polynucleotide, the polynucleotide and labeling moiety are bound via a spacer that can suppress or stop the progress of the DNA polymerase reaction so that the portion is not double-stranded together with the polynucleotide by the nucleic acid amplification reaction. Such a "spacer" may be any that can inhibit or stop the progress of the DNA polymerase reaction and prevent the labeling moiety from becoming double-stranded, and examples thereof include, but are not limited to, nucleic acid sequences with strong hairpin structures or pseudoknot structures, L-nucleic acids, peptide nucleic acids (PNAs), bridged nucleic acids (Bridged Nucleic Acids (BNAs) or Locked Nucleic Acids (LNAs)), fluorescein, Cy3, Cy5, divalent groups containing an azobenzene structure represented by formula I below, aliphatic chains (alkylene chains or polyoxyalkylene chains), and divalent groups containing an inverted sequence structure such as a 5'-5' bond or a 3'-3' bond.

[0119] [ka]

[0120] When two polynucleotide molecules are connected via a divalent group represented by Formula I, the phosphate group at the 3' end of one of the divalent groups refers to the phosphate group of the 5'-terminal nucleotide of one polynucleotide molecule, and the oxygen atom at the 5' end of the other divalent group forms a phosphate ester bond with the phosphate group of the 3'-terminal nucleotide of the other polynucleotide molecule.

[0121] Examples of the aliphatic chain spacer include a spacer represented by the following formula (II): 5'-OC m H 2m -O-3' Formula (II) (In the formula, 5' represents the oxygen atom of the 5'-side phosphodiester bond, 3' represents the oxygen atom of the 3'-side phosphodiester bond, and m represents an integer of 1 or more and 40 or less. H may be substituted with a substituent.)

[0122] In formula (II), m is preferably 2 or more and 36 or less, more preferably 3 or more and 16 or less. H in formula (II) may be substituted with a substituent, and typical examples of the substituent include an alkyl group, an alkoxy group, and a hydroxyl group. The alkyl group and alkoxy group as the substituent preferably have 1 to 8 carbon atoms, more preferably 1 to 4 carbon atoms. When two or more substituents are present, the substituents may be the same or different. Furthermore, it is also preferable that the compound has no substituent.

[0123] Other spacers include spacers represented by the following formula (III). 5'-(OC n H 2n ) L -O-3' Formula (III) (In the formula, 5' represents the oxygen atom of the 5'-side phosphodiester bond, 3' represents the oxygen atom of the 3'-side phosphodiester bond, n represents an integer of 2 or more and 4 or less, L represents an integer of 1 or more such that (n+1)×L is 40 or less, and H may be substituted with a substituent.)

[0124] In formula (III), (n+1)×L is preferably 2 or more and 36 or less, more preferably 3 or more and 16 or less. The same embodiments as those for the substituents in formula (II) are applicable to the substituents of H in formula (III).

[0125] Other aliphatic chain spacers include, for example, the following divalent groups:

[0126] [ka]

[0127] When two polynucleotide molecules are connected via these divalent groups, the phosphate group at one end of each divalent group points to the phosphate group of the 3'- or 5'-terminal nucleotide of one polynucleotide molecule, and the oxygen atom at the other end forms a phosphate ester bond with the phosphate group of the 5'- or 3'-terminal nucleotide of the other polynucleotide molecule.

[0128] (Binding moiety and solid support) The binding moiety is a tag capable of binding to a solid phase carrier, which will be described later. In this specification, a tag capable of binding to a solid phase carrier may be referred to as an immobilization tag.

[0129] The immobilization tag that can be used as a binding moiety is not particularly limited as long as it can bind to a solid-phase support and can be selected appropriately depending on the structure of the solid-phase support. For example, a polynucleotide (DNA, RNA, etc.), a protein, a peptide, or other compound (e.g., a low-molecular-weight compound), or a combination thereof can be used. The immobilization tag preferably contains or consists of a polynucleotide. The polynucleotide that can be contained in the immobilization tag is not particularly limited as long as it does not substantially interfere with the nucleic acid amplification reaction using the primer set. It is preferably a polynucleotide having, for example, 5 to 50 bases, preferably 10 to 35 bases, and more preferably a polynucleotide containing (or consisting of) the base sequence shown in SEQ ID NO: 14 or 15, or a partial base sequence thereof, or a complementary base sequence thereof.

[0130] The solid phase carrier is not particularly limited, and can be made of resin, metal, polysaccharide, mineral, etc., and can be in the form of a membrane, film, nonwoven fabric, plate, gel, etc. Preferably, the solid phase carrier has a porous structure that allows the amplification product and labeling substance in solution to develop. Examples of solid phase carriers that can be used in the present invention include filter paper, nitrocellulose membrane, polyethersulfone membrane, nylon membrane, various dried gels (silica gel, agarose gel, dextran gel, gelatin gel), silicon, glass, plastic, etc. The size and shape of the solid phase carrier can be appropriately selected to suit various operations and detections.

[0131] The solid phase carrier may be configured so that at least a portion thereof can bind to the immobilization tag, and more preferably, only a portion thereof can bind to the immobilization tag. By configuring only a specific portion of the solid phase carrier to be capable of binding to the immobilization tag, the amplification product captured on the solid phase carrier is detected only in that portion, making it easier to determine whether the result is positive or negative.

[0132] The binding between the solid-phase carrier and the immobilization tag may be a direct binding or an indirect binding, and the binding means can be appropriately selected according to the combination of the solid-phase carrier and the immobilization tag used. For example, when the immobilization tag contains a polynucleotide, a polynucleotide capable of hybridizing to the polynucleotide (for example, a polynucleotide containing a sequence complementary to the base sequence of the polynucleotide) is immobilized on the solid-phase carrier as a tag capture means, and the two polynucleotides are hybridized to indirectly bind the solid-phase carrier and the immobilization tag. The immobilization of the polynucleotide on the solid-phase carrier may be carried out via a peptide, protein, nucleic acid, etc., or via an appropriate functional group. The hybridization conditions can be carried out according to the conditions described above for the binding between the labeling tag and the labeling substance. When immobilizing a polynucleotide on a solid-phase carrier, by immobilizing it in a limited specific part, the captured amplification product is detected only in a predetermined part, so that the discrimination of positive or negative can be facilitated.

[0133] The binding part (immobilization tag) and the polynucleotide contained in the first primer or the polynucleotide contained in the second primer can be bound by any means, and can be directly or indirectly bound. However, when at least the part connecting to the polynucleotide of the immobilization tag consists of a polynucleotide, the polynucleotide and the immobilization tag are bound via a spacer capable of suppressing or stopping the progress of the DNA polymerase reaction so that the part is not double-stranded with the polynucleotide during the nucleic acid amplification reaction. Specific examples of the spacer provided between the binding part and the polynucleotide are the same as those described above for the spacer provided between the labeling part and the polynucleotide.

[0134] <Method for determining genotype using a primer set for detecting an index base sequence specific to C28 strain> Using the primer set according to the third embodiment, which includes the first and second primers, it is possible to determine whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine.

[0135] This determination method is, for example, carrying out a nucleic acid amplification reaction using the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined as a template and the primer set according to the third embodiment, which includes the first primer and the second primer; Detecting an amplification product from the nucleic acid amplification reaction; and determining that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine when the amplification product is detected, and determining that the base is thymine when the amplification product is not detected; Includes.

[0136] The primer set according to the third embodiment, which includes the first and second primers, can also be used in a method for determining whether the base corresponding to position 28 in the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium is cytosine. carrying out a nucleic acid amplification reaction using the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined as a template and the primer set according to the third embodiment, which includes the first primer and the second primer; Detecting an amplification product from the nucleic acid amplification reaction; and determining that the base in the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine when the amplification product is detected; The method can include:

[0137] Here, "polynucleotides derived from the genomic DNA of the acid-fast bacterium to be evaluated" include partial polynucleotides of a portion of the genomic DNA, and amplification products obtained by amplifying the entire genomic DNA or a portion containing the indicator base sequence by a nucleic acid amplification reaction. Examples of nucleic acid amplification reactions are as described above.

[0138] If the genomic DNA of the mycobacterium to be evaluated or a polynucleotide derived from the genomic DNA of the mycobacterium to be evaluated contains a target sequence obtained by a nucleic acid amplification reaction using the first primer and the second primer, a polynucleotide fragment containing the target sequence will be produced as the amplification product.

[0139] The method for detecting the amplification products produced by the nucleic acid amplification reaction is not particularly limited, and examples thereof include a method in which the reaction solution of the nucleic acid amplification reaction is fractionated by gel electrophoresis and the presence or absence of a band of a size corresponding to a polynucleotide fragment containing a predetermined target sequence is confirmed, and a method in which a labeled polynucleotide probe specific to a polynucleotide fragment containing a predetermined target sequence is hybridized to the reaction solution of the nucleic acid amplification reaction or its product, and the complex is detected.

[0140] Another embodiment of the method for detecting an amplification product is to carry out a nucleic acid amplification reaction in the presence of a probe according to the second embodiment of the present invention, in which a reporter fluorescent substance is linked to the 5' end of a polynucleotide and a quencher dye is linked to the 3' end, and detect the polynucleotide fragment using, as an indicator, the fluorescence generated when a polynucleotide fragment containing a predetermined target sequence is amplified.

[0141] Furthermore, when one of the first primer and the second primer further comprises a labeling portion that is a tag or a labeling substance that can bind to a labeling substance, and the other further comprises a binding portion that is a tag that can bind to a solid phase carrier, the following detection step using a solid phase carrier can be carried out.

[0142] (Detection step using a solid phase carrier) In this detection step, the product of the nucleic acid amplification reaction is brought into contact with a solid phase carrier containing, at least in part, a portion capable of binding to the binding moiety, and the amplified product at the portion of the solid phase carrier is detected using the labeling moiety as an indicator.

[0143] When the labeling moiety is the above-mentioned labeling tag, a labeling step of binding a labeling substance to the labeling tag may be further carried out. Details of the labeling step will be described later. When the labeling moiety is the above-mentioned labeling substance, the labeling step is not necessary. In the detection step, "using the labeling moiety as an indicator" refers to detecting the amplification product using the labeling substance bound through the labeling step as an indicator when the labeling moiety is a labeling tag, and refers to detecting the amplification product using the labeling substance as an indicator when the labeling moiety is a labeling substance.

[0144] The product of the nucleic acid amplification reaction refers to the reaction solution of the nucleic acid amplification reaction which may contain the amplification product, or a sample obtained by further increasing the concentration of the amplification product from the reaction solution. The details of the solid phase carrier are as described above.

[0145] The contact of the product with the binding moiety of the solid phase carrier and the binding moiety can be carried out under conditions (e.g., hybridization conditions or buffer conditions of about pH 5 to 9) that are appropriately adjusted depending on the combination of the solid phase carrier and the binding moiety so that, if an amplification product is contained in the product, the binding moiety of the amplification product will bind to the binding moiety.

[0146] The amplification product can be detected by detecting, preferably visually detecting, the labeling substance bound to the amplification product captured on the solid-phase carrier. If the amplification product is present, the labeling substance of the amplification product captured and bound to the solid-phase carrier is detected. The presence or absence of the detection can be used as an indicator to determine the presence or absence of an amplification product (a polynucleotide fragment containing a target sequence) in the reaction system of the nucleic acid amplification reaction.

[0147] (labeling process) The labeling step is a step performed when the labeling moiety included in the primer set is the above-mentioned labeling tag, and is performed by contacting the product of the nucleic acid amplification reaction with a labeling substance and binding the labeling substance to the labeling tag. The labeling step may be performed before, after, or simultaneously with contacting the product of the nucleic acid amplification reaction with the solid phase support.

[0148] The contact between the labeling substance and the product may be carried out under conditions (for example, the hybridization conditions described above or buffer conditions with a pH of about 5 to 9) that are appropriately adjusted depending on the combination of the labeling substance and the labeling tag so that, if an amplification product is contained in the product, the labeling substance will bind to the labeling tag of the amplification product.

[0149] (detection device) The above-mentioned detection and labeling steps can be performed using a nucleic acid detection device that utilizes nucleic acid chromatography. By using this nucleic acid detection device, the presence or absence of an amplification product (a polynucleotide fragment containing a target sequence) in a nucleic acid amplification reaction system can be detected and determined without the need for a special device, and results can be obtained easily and quickly.

[0150] The nucleic acid detection device can be a known nucleic acid detection device (WO2012 / 070618) that is used to detect labeled nucleic acid amplification products by nucleic acid chromatography.

[0151] A schematic diagram of one embodiment of a nucleic acid detection device that can be used in the present invention is shown in Figure 1, but the nucleic acid detection device is not limited to this embodiment. In the following description, the reference numerals assigned to the respective components correspond to the reference numerals shown in Figure 1.

[0152] The nucleic acid detection device 10 of FIG. 1 is formed by arranging a sample pad 3, which is a reaction system receiving portion for receiving a reaction system for a nucleic acid amplification reaction, a conjugate pad 2 for holding a labeled substance, a porous solid-phase carrier 1 including a portion 6 capable of binding to a binding moiety contained in the amplification product, and an absorbent pad 4 in contact with each other in this order on a substrate 5. The portion 6 of the solid-phase carrier 1 is a portion where a means for capturing the amplification product (capture means) (e.g., the above-mentioned oligonucleotide, etc.) is locally arranged and immobilized. Although not shown, the surface of the solid-phase carrier 1 may be covered with a film. The sample pad 3, conjugate pad 2, solid-phase carrier 1, and absorbent pad 4 can be composed of a material having a porous structure that can be used as the solid-phase carrier. These may be composed of the same material or different materials. The substrate 5 can support various components arranged thereon and facilitate operation of the nucleic acid detection device. For example, a material made of resin, metal, mineral, etc. can be used. When a labeled substance is mixed into the developing solution or when the labeled portion is a labeled substance, the conjugate pad 2 can be omitted.

[0153] The reaction system for nucleic acid amplification reaction is added to the sample pad 3. The reaction system may be added as is, or may be added together with an appropriate developing solution (e.g., phosphate buffer, Tris buffer, Good's buffer, SSC buffer). The developing solution may further contain surfactants, salts, proteins, nucleic acids, etc., as needed. The reaction system added to the sample pad 3 develops from upstream to downstream by capillary action in the direction indicated by the arrow in Figure 1.

[0154] As another aspect, the nucleic acid detection device can be placed in a container (e.g., a PCR tube, an Eppendorf tube, a 96-well plate, etc.) holding the product of the nucleic acid amplification reaction and / or the developing solution, and developed by a method of immersing the sample pad 3 in the product of the nucleic acid amplification reaction and / or the developing solution. In that case, the width of the sample pad 3 is preferably 2.0 to 10.0 mm, more preferably 2.0 to 5.0 mm, so that the sample pad 3 can enter the container holding the product of the nucleic acid amplification reaction and / or the developing solution.

[0155] In the embodiment where the labeling part is a labeling tag, when the amplification product in the reaction system passes through the conjugate pad 2 holding the labeling substance, it contacts the labeling substance and is labeled by the labeling substance via the labeling tag. Next, when the amplification product in the reaction system passes through the solid-phase carrier 1, it contacts the capture means fixed to the part 6 and is captured and bound to the solid-phase carrier 1 via the immobilization tag.

[0156] When the amplification product is present, the labeling substance bound to the amplification product captured and bound to the part 6 of the solid-phase carrier 1 containing the capture means is detected in the part 6. If the labeling substance can be visually confirmed, the part 6 will be colored due to the labeling substance. Based on the presence or absence of the detection (color development) of the labeling substance, the presence or absence of the amplification product in the sample can be discriminated.

[0157] <Method for determining susceptibility to macrolide antibiotics using a primer set for detecting an index base sequence specific to C28 strain> Using the primer set according to the third embodiment including the first primer and the second primer, the susceptibility of acid-fast bacteria, particularly acid-fast bacteria belonging to the Mycobacterium abscessus complex, to macrolide antibiotics can be determined.

[0158] This determination method is, for example, carrying out a nucleic acid amplification reaction using the genomic DNA of the acid-fast bacterium to be determined or a polynucleotide derived from the genomic DNA of the acid-fast bacterium to be determined as a template and the primer set according to the third embodiment, which includes the first primer and the second primer; Detecting an amplification product from the nucleic acid amplification reaction; and determining that the acid-fast bacterium is susceptible to macrolide antibiotics when the amplification product is detected, and determining that the acid-fast bacterium is not susceptible to macrolide antibiotics when the amplification product is not detected; Includes.

[0159] The nucleic acid amplification reaction and detection of the amplified product in the determination method can be carried out in the same manner as described above.

[0160] <Method 2 for determining the erm(41) genotype of mycobacteria> A fourth embodiment of the present invention is 1. A method for determining whether the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine, comprising: An indicator base sequence is detected in the genomic DNA of the acid-fast bacterium to be evaluated, the indicator base sequence being present in a region of the genomic DNA other than the erm(41) gene when the base of the erm(41) gene is thymine, and not present when the base is cytosine. Including, detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is thymine; The absence of detection of the indicator base sequence indicates that the base in the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine. Regarding the method. [Example]

[0161] The genome information of the reference strain of Mycobacteroides abscessus complex was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). The genome information of the clinical isolates and publicly available strains of Mycobacteroides abscessus complex (listed in Table 5 below) was obtained by analyzing DNA prepared from pure cultures using a next-generation sequencer (MiSeq (Illumina)). Based on this genome information, we performed molecular phylogenetic analysis of strains with a normal (T28) erm(41) gene (T28 strain) and strains with a mutant (C28) erm(41) gene (C28 strain). We found that the T28 and C28 strains can be classified into two distinct molecular phylogenetic groups. Furthermore, we performed comparative analysis of the nucleic acid sequences using Mauve software (Darling et al., 2005) and found that the C28 strain possesses a highly conserved insertion sequence of SEQ ID NO:2. The base sequence of SEQ ID NO: 2 may be referred to as the "inserted base sequence."

[0162] Example 1 C28-f primer and C28-r primer were designed as a primer set that specifically hybridizes to the inserted nucleotide sequence of SEQ ID NO: 2. The sequences of each primer are shown in Table 2. PCR was performed using the designed primer set, and the nucleic acid amplification products were analyzed by nucleic acid chromatography to verify whether the C28 strain could be specifically detected.

[0163] (I) Primer structure To enable detection by nucleic acid chromatography, DNA consisting of the following tag sequence was linked to the 5' end of each primer via a divalent group represented by formula I above, which contains an azobenzene structure that inhibits polymerase reaction. Here, a phosphate ester bond is formed between the 5' end of the divalent group containing the azobenzene structure shown in formula I above and the phosphate group of the 3'-terminal nucleotide of the DNA consisting of the tag sequence, and a phosphate ester bond is also formed between the phosphate group at the 3' end of the divalent group and the hydroxyl group at the 5'-position of deoxyribose in the 5'-terminal nucleotide of the primer portion of the primer.

[0164] An immobilization tag 1 consisting of the base sequence of SEQ ID NO: 15 was attached to the 5' end of the C28-f primer consisting of the base sequence of SEQ ID NO: 12 via an azobenzene structure. Similarly, a labeling tag 1 consisting of the base sequence of SEQ ID NO: 14 was attached to the 5' end of the C28-r primer consisting of the base sequence of SEQ ID NO: 13.

[0165] A combination of tagged C28-f and C28-r primers was used as the first primer set.

[0166] The base sequence of SEQ ID NO:12 is a partial base sequence from positions 4634 to 4658 of SEQ ID NO:2, and is a partial base sequence from positions 581 to 605 of SEQ ID NO:7.

[0167] The base sequence of SEQ ID NO: 13 is a complementary base sequence to the partial base sequence from positions 4704 to 4730 of SEQ ID NO: 2, and is a complementary base sequence to the partial base sequence from positions 651 to 677 of SEQ ID NO: 7.

[0168] [Table 2]

[0169] (II) Preparation of oligonucleotide-conjugated gold colloids Gold colloid (Gold Colloid, 40 nm, 9.0 × 10 10(particle number / ml, British Biocell International) was mixed with a thiol group-containing oligonucleotide having the base sequence of SEQ ID NO: 16 in Table 3 and incubated at 50°C for 16 hours. After centrifugation at 6000 rpm for 15 minutes and removal of the supernatant, 0.05 M sodium chloride and 5 mM phosphate buffer (pH 7) were added and mixed, followed by incubation again at 50°C for 40 hours.

[0170] After incubation, the mixture was centrifuged (6000 rpm, 15 minutes), the supernatant was removed, and 5 mM phosphate buffer (pH 7) was added. This buffer replacement was repeated. By the above procedures, oligonucleotide-bound gold colloid was prepared.

[0171] The prepared suspension of oligonucleotide-conjugated gold colloid was added uniformly to a glass fiber pad, and then dried in a vacuum dryer to obtain a conjugate pad.

[0172] (Thiol-containing oligonucleotides) The thiol group-containing oligonucleotide is an oligonucleotide having the base sequence of SEQ ID NO: 16, and a phosphate group at the 3' position of the 3' end of the oligonucleotide, HO-(CH2) m It is bonded to the hydroxyl group of a compound represented by -SH (m is 6) through a phosphate ester bond.

[0173] (III) Preparation of membrane with immobilized tag capture means A solution containing oligonucleotide probe 1 consisting of the base sequence of SEQ ID NO: 17 as a tag capture means was applied to a nitrocellulose membrane (Hi-Flow180) manufactured by Merck Millipore using a dispenser in the form of a 1 mm wide line perpendicular to the direction of development. The membrane was then dried at 40°C for 30 minutes to obtain a tag capture means-equipped membrane. The line coated with oligonucleotide probe 1 of SEQ ID NO: 17 was designated as the T1 line.

[0174] [Table 3]

[0175] (IV) Fabrication of nucleic acid detection device A nucleic acid detection device was produced in accordance with the detection device shown in the schematic diagram of FIG. That is, a polypropylene backing sheet (Lohmann) was used as the substrate 5, the conjugate pad prepared in (II) above was used as the conjugate pad 2, the membrane (solid phase carrier) 1 prepared in (III) above had an oligonucleotide probe 1 provided in portion 6 as a tag capture means, a glass fiber sample pad was used as the sample pad 3, and a cellulose absorbent pad was used as the absorbent pad 4, all of which were stacked and bonded together as shown in Figure 1 to produce a nucleic acid detection device 10.

[0176] (V)PCR Using the tagged first primer set described in (I) above, the following PCR reaction solution was prepared according to the TaKaRa TaqHS perfect Mix manual.

[0177] [Table 4]

[0178] The template DNA used was DNA purified from the mycobacterial strains shown in Table 5 (Mycobacteroides abscessus complex and other clinical isolates, publicly available strains, and closely related mycobacterial strains). As a negative control, a PCR reaction solution with the same composition was prepared, except that 1 μL of sterile distilled water was used instead of the template DNA solution. The 5 μM Fw primer solution and 5 μM Rv primer solution were prepared by dissolving the designated primers in sterile distilled water to a concentration of 5 μM.

[0179] The PCR reaction solution was placed in a PCR device (Bioer, LifeEco) and reacted at 94°C for 1 minute, followed by 35 cycles of 94°C for 5 seconds / 62°C for 10 seconds / 72°C for 5 seconds.

[0180] As mentioned above, each of the Mycobacteroides abscessus complex strains used in the test was identified by sequence analysis as either the T28 strain or the C28 strain.

[0181] (VI) Detection using a nucleic acid chromatography detection system The reaction solution after PCR under the above conditions was applied to the nucleic acid detection device 10 to attempt detection of the nucleic acid amplification product. Specifically, 5 μL of the reaction solution after PCR was added to the sample pad 3 on the device 10, and then 80 μL of a developing solution (citrate buffer containing a surfactant) was added to develop the reaction solution. After 10 minutes at room temperature, the presence or absence of coloration of the T1 line in the portion 6 containing the tag capture means immobilized in a line on the membrane 1 was visually confirmed.

[0182] If coloration of the T1 line is confirmed, it indicates that the inserted base sequence shown in SEQ ID NO: 2, which is specifically present in the C28 strain, was contained in the template DNA, and that a nucleic acid amplification product derived from the C28 strain was obtained. The results are shown in Table 5 (Table 5-1 and Table 5-2) and Figure 2. In Table 5, when coloring was confirmed, it was indicated as "+", and when it was not confirmed, it was indicated as "-".

[0183] [Table 5-1] [Table 5-2]

[0184] As a result of the test, it was confirmed that the constructed chromatographic detection system can specifically detect the Mycobacteroides abscessus complex strain (C28 strain) in which the 28th position of the erm(41) gene is cytosine, and there is no cross-reaction with the T28 strain or other mycobacterial species.

[0185] <Example 2> Primers for specific detection of the C28 strain were designed based on the sequence information of the insert sequence (SEQ ID NO: 2) that is specifically present in the genomic DNA of the C28 strain. Each primer was designed to specifically hybridize to a partial base sequence in the insert sequence and to trigger a nucleic acid amplification reaction when C28 strain-derived DNA was present in the sample. PCR was performed using the designed primers, and the nucleic acid amplification products were analyzed by agarose gel electrophoresis to verify whether the C28 strain could be specifically detected.

[0186] [Table 6]

[0187] (I) PCR The following PCR reaction solution was prepared according to the TaKaRa TaqHS perfect Mix manual using the second primer set consisting of SEQ ID NOs: 18 and 19, the third primer set consisting of SEQ ID NOs: 20 and 21, the fourth primer set consisting of SEQ ID NOs: 22 and 23, the fifth primer set consisting of SEQ ID NOs: 24 and 25, the sixth primer set consisting of SEQ ID NOs: 26 and 27, and the seventh primer set consisting of SEQ ID NOs: 28 and 29, all of which are listed in Table 6.

[0188] [Table 7]

[0189] Purified genomic DNA prepared from Mycobacteroides abscessus ATCC19977 and Mycobacteroides abscessus LRC18036 was used as template DNA. Mycobacteroides abscessus ATCC19977 is a T28 strain, and Mycobacteroides abscessus LRC18036 is a C28 strain. Based on the nucleotide sequence information, PCR using the second primer set and the genomic DNA of Mycobacteroides abscessus LRC18036 as a template is predicted to specifically amplify a 336-bp region. PCR using the third primer set is predicted to specifically amplify a 940-bp region. PCR using the fourth primer set is predicted to specifically amplify a 1040-bp region. PCR using the fifth primer set is predicted to specifically amplify a 51-bp region. It is estimated that if PCR is performed in the same manner using the sixth primer set, a 479 bp region will be specifically obtained as a nucleic acid amplification product. It is estimated that if PCR is performed in the same manner using the seventh primer set, a 698 bp region will be specifically obtained as a nucleic acid amplification product. On the other hand, if PCR is performed using the genomic DNA of Mycobacteroides abscessus ATCC19977, a T28 strain, as a template, it is estimated that no nucleic acid amplification product will be obtained using any of the second to seventh primer sets.

[0190] As a negative control, a PCR reaction mixture was prepared with the same composition as above, except that 1 μL of sterile distilled water was used instead of the template DNA solution. The 5 μM Fw primer solution and 5 μM Rv primer solution were prepared by dissolving the designated primers in sterile distilled water to a concentration of 5 μM.

[0191] The PCR reaction mixture was placed in a PCR device (Bioer, LifeEco) and incubated at 94°C for 1 minute, followed by 35 cycles of 94°C for 5 seconds, 62°C for 10 seconds, and 72°C for 5 seconds. The PCR reaction mixture was then subjected to agarose gel electrophoresis to detect the presence or absence of nucleic acid amplification products. The results are shown in Figure 3. In Figure 3, A represents the results of agarose gel electrophoresis of the PCR amplification products using the second primer set, B the third primer set, C the fourth primer set, D the fifth primer set, E the sixth primer set, and F the seventh primer set.

[0192] As a result of the test, regardless of which primer set was used, specific nucleic acid amplification products were confirmed by agarose gel electrophoresis analysis only when DNA derived from the C28 strain (Mycobacteroides abscessus LRC18036) of the Mycobacteroides abscessus complex was contained in the sample. These results confirmed that the C28 strain can be specifically detected by detecting the presence or absence of the inserted base sequence shown in SEQ ID NO: 2. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for determining whether a base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in an erm(41) gene of an acid-fast bacterium belonging to the Mycobacteroides abscessus complex is cytosine or thymine, comprising: An indicator base sequence is detected in the genomic DNA of the acid-fast bacterium to be evaluated, the indicator base sequence being present in a region of the genomic DNA other than the erm(41) gene when the base of the erm(41) gene is cytosine, and not present when the base is thymine. Including, detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is cytosine; the absence of detection of the indicator base sequence indicates that the base of the erm(41) gene in the genomic DNA of the acid-fast bacterium is thymine; the index base sequence is a first base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 2 or a second base sequence complementary to the first base sequence; method.

2. The method according to claim 1, wherein the first base sequence comprises at least a portion of a base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11.

3. A primer set for detecting an indicator base sequence that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene is cytosine in an acid-fast bacterium belonging to the Mycobacteroides abscessus complex, and that is absent when the base is thymine, a first primer containing a polynucleotide containing, at its 3' end, a base sequence f12 that can hybridize with a complementary base sequence of a partial base sequence f11 of 15 or more consecutive bases contained in the index base sequence; a second primer containing a polynucleotide containing, at its 3' end, a base sequence r12 that is contained in the index base sequence and is located closer to the 3' end than the 3' end of the partial base sequence f11 and that is capable of hybridizing to a partial base sequence r11 of 15 or more consecutive bases; Including, the index base sequence is a third base sequence of 40 or more consecutive bases contained in the base sequence of SEQ ID NO: 2; Primer set.

4. The primer set according to claim 3, wherein the base sequence from the 5'-terminal base of the partial base sequence f11 to the 3'-terminal base of the partial base sequence r11 contained in the third base sequence contains at least a portion of a base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11.

5. The partial base sequence f11 is (f11-12-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2; (f11-18-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2; (f11-20-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2; (f11-22-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2; (f11-24-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2; (f11-26-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2, or (f11-28-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 and The partial base sequence r11 is (r11-13-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2; (r11-19-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2; (r11-21-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2; (r11-23-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2; (r11-25-1) a base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2; (r11-27-1) A base sequence of 10 or more consecutive bases included in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2, or (r11-29-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 That is, The primer set according to claim 3 or 4.

6. The primer set according to any one of claims 3 to 5, wherein one of the first primer and the second primer further comprises a labeling moiety that is a tag or a labeling substance that can bind to a labeling substance, and the other primer further comprises a binding moiety that is a tag that can bind to a solid phase carrier.

7. The primer set according to claim 6 ; and a solid phase carrier at least partially including a portion capable of binding to the binding portion. A kit for detecting an indicator base sequence in an acid-fast bacterium belonging to the Mycobacteroides abscessus complex, which is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene is cytosine, but is not present when the base is thymine.

8. A probe for detecting an indicator base sequence that is present in a region of genomic DNA other than the erm(41) gene when the base corresponding to position 28 of the base sequence of SEQ ID NO: 1 in the erm(41) gene is cytosine in an acid-fast bacterium belonging to the Mycobacteroides abscessus complex, and that is absent when the base is thymine, a polynucleotide containing a base sequence capable of hybridizing with a partial base sequence p1 of 15 or more consecutive bases contained in the index base sequence; the index base sequence is a first base sequence of 20 or more consecutive bases contained in the base sequence of SEQ ID NO: 2 or a second base sequence complementary to the first base sequence; probe.

9. 9. The probe according to claim 8, wherein the partial base sequence p1 at least partially comprises a base sequence of 10 or more consecutive bases contained in the base sequence of SEQ ID NO: 3, 5, 7, 9 or 11 or a complementary base sequence thereof.

10. The partial base sequence p1 is (p1-12-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4624 to 4668 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-18-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2356 to 2397 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-20-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2624 to 2663 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-22-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4044 to 4083 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-24-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 6535 to 6575 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-26-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7478 to 7520 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-28-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7629 to 7673 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-13-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 4694 to 4740 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-19-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 2672 to 2711 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-21-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 3539 to 3583 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-23-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 5061 to 5103 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-25-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 6566 to 6605 of the base sequence of SEQ ID NO: 2 or its complementary base sequence; (p1-27-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 7931 to 7976 of the base sequence of SEQ ID NO: 2 or its complementary base sequence, or (p1-29-1) A base sequence of 10 or more consecutive bases contained in the base sequence ranging from positions 8302 to 8346 of the base sequence of SEQ ID NO: 2 or its complementary base sequence The probe according to claim 8 or 9,

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