JP7575101B2 - Method for detecting mutant bacteria resistant to macrolide antibiotics - Google Patents

Description

The present invention relates to a method for detecting mutant bacteria resistant to macrolide antibiotics. In particular, the present invention relates to a method for detecting mutant bacteria resistant to macrolide antibiotics, comprising detecting point mutations of two adenines to which macrolide antibiotics bind in the 23S rRNA domain V region of the macrolide antibiotic-resistant mutant bacteria by PCR amplification. In particular, the present invention relates to a method for detecting drug-resistant bacteria of Mycoplasma pneumoniae , comprising detecting point mutations of adenines at positions 2063 and 2064 in the 23S rRNA domain V region of Mycoplasma pneumoniae. In particular, the present invention relates to a method for detecting macrolide-resistant bacteria of Bordetella pertussis , comprising detecting point mutations of adenines at positions 2037 and 2038 in the 23S rRNA domain V region of Bordetella pertussis.

Macrolide antibiotics are considered the first or primary choice for the treatment of infections caused by Mycoplasma pneumoniae , Bordetella pertussis , Helicobacter pylori , Campylobacter jejuni , Chlamydia trachomatis , Mycobacterium avium , Mycobacterium intracellulare , and other bacteria.

Macrolide antibiotics bind to domain V of 23S rRNA, inhibiting its function and suppressing protein synthesis, thereby acting as antibiotics. The important sites for macrolide antibiotics to bind to domain V are two adjacent adenines at base numbers 2058 and 2059 in E. coli. If base substitution or methylation mutation occurs at these sites, the three-dimensional structure changes, and macrolide antibiotics are no longer able to bind to domain V and inhibit protein synthesis, resulting in the bacteria acquiring resistance to macrolide antibiotics to a degree that becomes clinically problematic. Therefore, if these sites can be detected and if it is known whether base substitution has occurred or not, bacteria can be detected and drug resistance can be determined.

The two adjacent adenines in domain V of 23S rRNA to which macrolide antibiotics bind are at positions 2058 and 2059 in Escherichia coli, which is the most widely studied bacterium, but are at positions 2063 and 2064 in Mycoplasma pneumoniae (type strain M129; NCBI Reference Sequence: NC_000912.1), 2037 and 2038 in Bordetella pertussis (type strain Tohama I; NCBI Reference Sequence: NC_002929.2), 2146 and 2147 in Helicobacter pylori (type strain Helicobacter pylori 26695; NCBI Reference Sequence: NC_000915.1) (some sources consider them to be positions 2142 and 2143, based on the sequence of Accession number U27270), and Campylobacter jejuni (type strain Campylobacter jejuni subsp. The positions 2074 and 2075 in Chlamydia trachomatis (type strain Chlamydia trachomatis D/UW-3/CX; NCBI Reference Sequence: NC_000117.1), 2038 and 2039 in Mycobacterium avium (type strain Mycobacterium avium 104; NCBI Reference Sequence: NC_008595.1), and 2266 and 2267 in Mycobacterium intracellulare (type strain Mycobacterium intracellulare ATCC 13950; NCBI Reference Sequence: NC_016946.1) correspond to the positions of two adjacent adenines in domain V of 23S rRNA to which macrolide antibiotics bind. Therefore, if these sites can be detected and it can be determined whether or not base substitution has occurred, it will be possible to detect the bacteria and determine drug resistance.

The most important mechanism for drug-resistant Mycoplasma pneumoniae, a mutant bacterium resistant to macrolide antibiotics, is a point mutation of adenine at positions 2063 and 2064 in the domain V region, which is the functional site of 23S rRNA. Conventional methods for detecting point mutations include (1) PCR-RFLP, (2) sequencing, and (3) Qprobe. All of these methods use a base sequence containing a mutation. In (1) PCR-RFLP, the product amplified by nested PCR is treated with a restriction enzyme, and the cleavage pattern is confirmed by electrophoresis. In (2) sequencing, the product is amplified by PCR, and the DNA base sequence of the amplified product is compared with the base sequence of a standard strain using BLAST. In (3) Qprobe, after amplification by PCR, a quenching probe is bound to and dissociated from the mutation site by temperature change, and the presence or absence of a mutation is confirmed using the difference in Tm value at the time of fluorescence emission.

Conventional methods for detecting drug-resistant Mycoplasma pneumoniae bacteria using a PCR method characterized by DNA polymerase are disclosed in Patent Document 1 and Non-Patent Document 1.
Patent Document 1 relates to a method for detecting the A2063G mutation (a mutation in which adenine (A) at position 2063 is replaced with guanine (G)) in 23S rRNA of Mycoplasma pneumoniae, and uses a primer with a modified 3' end and Q5 High-Fidelity DNA polymerase. Q5 High-Fidelity DNA polymerase is a DNA polymerase introduced by New England Biolabs (NEB) that is characterized by its high accuracy and amplificability independent of GC content.

Non-patent literature 1 relates to a detection method using High-Fidelity DNA polymerase and 3' phosphorothioate-modified specific primers developed to assay 23S rRNA gene mutation sites (mainly at positions 2063, 2064 and 2617) of Mycoplasma pneumoniae.

Both of the high-fidelity DNA polymerases described in Patent Document 1 and Non-Patent Document 1 recognize primers with a difference of one base at the 3' end, and are different from DNA polymerases in which the amplification efficiency is significantly reduced in the presence of a mismatch pair. In addition, the primers described in Patent Document 1 and Non-Patent Document 1 require special modification, and the methods described in Patent Document 1 and Non-Patent Document 1 cannot be said to be capable of inexpensively and easily detecting drug-resistant Mycoplasma pneumoniae bacteria.

CN107254524A

Liu, Yan et al., ZhongguoRenshouGonghuanbingXuebao(2015),31(5), 479-484

In the detection of mutant bacteria resistant to macrolide antibiotics, particularly in the detection of drug-resistant bacteria of Mycoplasma pneumoniae or Bordetella pertussis, there is a demand for a method that can detect the bacteria and distinguish between drug resistance at low cost, accurately, and in a short time. Standard clinical test methods for diagnosing Mycoplasma pneumoniae include culture methods, serological methods, and genetic testing methods. Of these, serological methods have problems in that they are insensitive and nonspecific for bacterial detection, and cannot distinguish the presence or absence of drug resistance.

Regarding genetic testing methods, the PCR-RFLP method has the disadvantage of requiring two rounds of base sequence amplification and restriction enzyme treatment, which makes the process complicated and involves many steps. The sequencing method has the disadvantage of taking time and effort to sequence and analyze the base sequence. The Qprobe method has the disadvantage of requiring a probe that is labeled with a fluorescent dye, fluoresces alone, and whose fluorescence decreases upon hybridization, and also requires an expensive real-time PCR fluorescence detection system. In addition, if the target site is not a known mutation, it may not be recognized correctly. For example, there are devices that use the Qprobe method to detect 23SrRNA mutations (fully automated genetic analyzer GENECUBE (registered trademark), unit price 16 million yen) and research reagents (Genecube (registered trademark) Test Helicobacter pylori 23SrRNA, 96 kits/150,000 yen), but these are expensive.

The present invention comprises:
1. A method for detecting a mutant bacterium resistant to a macrolide antibiotic, comprising detecting a point mutation of two adenines to which a macrolide antibiotic binds in the V region of the 23S rRNA domain of the mutant bacterium resistant to a macrolide antibiotic by PCR amplification using a DNA polymerase that recognizes a primer having a difference of one base at the 3' end and in which the amplification efficiency is significantly reduced in the presence of a mismatch pair.
2. The method according to the preceding item 1, wherein the macrolide antibiotic-resistant mutant bacterium is a drug-resistant bacterium of Mycoplasma pneumoniae, and the two adenines to which the macrolide antibiotic binds in the 23S rRNA domain V region are at positions 2063 and 2064 in the 23S rRNA domain V region of Mycoplasma pneumoniae (standard strain M129; NCBI Reference Sequence: NC_000912.1).
3. The method according to the preceding item 1, wherein the macrolide antibiotic-resistant mutant bacterium is a drug-resistant bacterium of Bordetella pertussis, and the two adenines to which the macrolide antibiotic binds in the 23S rRNA domain V region are located at positions 2037 and 2038 in the 23S rRNA domain V region of Bordetella pertussis (standard strain Tohama I; NCBI Reference Sequence: NC_002929.2).
4. The method according to the preceding item 1 or 2, wherein the DNA polymerase is HiDi DNA polymerase (registered trademark).
5. Reverse primers for detecting adenine mutations at positions 2063 and 2064 of the 23S rRNA sequence of M. pneumoniae in PCR amplification:
- a primer having a length of 20 to 35 bases and containing the sequence shown in 5'-GTAAAGCTTCACGGGGTCTC-3' on the 3'side;
5. The method according to claim 2 or 4, which uses a 20- to 35-base-long primer containing the sequence shown in 5'-TAAAGCTTCACGGGGTCTTT-3' on its 3' side, and a 20- to 35-base-long primer containing the sequence shown in 5'-TAAAGCTTCACGGGGTCTTC-3' on its 3' side.
6. A reverse primer consisting of the sequence shown in 5'-GCTACAGTAAAGCTTCACGGGGTCTC-3' (SEQ ID NO: 1);
6. The method according to item 5 above, wherein the primer is a primer consisting of the sequence shown in 5'-TTAAGCTACAGTAAAGCTTCACGGGGTCTTT-3' (SEQ ID NO: 2), and the primer is a primer consisting of the sequence shown in 5'-TTAAGCTACAGTAAAGCTTCACGGGGTCTTC-3' (SEQ ID NO: 3).
7. Reverse primer for detecting the adenine mutations at positions 2037 and 2038 of the 23S rRNA sequence of Bordetella pertussis in PCR amplification:
A primer having a length of 20 to 35 bases and including a sequence shown in any one of SEQ ID NOs: 5 to 11 on the 3'side;
5. The method according to item 3 or 4 above, which uses a primer having a length of 20 to 35 bases and including a sequence shown in any one of SEQ ID NOs: 12 to 18 on the 3' side, and a primer having a length of 20 to 35 bases and including a sequence shown in any one of SEQ ID NOs: 19 to 25 on the 3' side.
8. The method according to item 7 above, wherein the reverse primer is a primer consisting of any one of the nucleotide sequences set forth in SEQ ID NOs: 5 to 11, a primer consisting of any one of the nucleotide sequences set forth in SEQ ID NOs: 12 to 18, and a primer consisting of any one of the nucleotide sequences set forth in SEQ ID NOs: 19 to 25.
9. The method according to any one of items 1 to 8 above, wherein the thermal cycler conditions are (1) 1 cycle at 98°C for 1 minute, followed by (2) 98°C for 10 seconds, (3) 72°C to 56°C for 15 seconds, and (4) 72°C for 30 seconds, for a total of 36 cycles consisting of (2) to (4), and then (5) 1 cycle at 72°C for 1 minute, in which the step (3) is always performed for 15 seconds, but the temperature in the first cycle starts at 72°C and is lowered by 1°C every cycle until the 16th cycle at 57°C, and the 17th to 36th cycles are performed at 56°C.

According to the present invention, in a laboratory environment where PCR can be performed, it is possible to easily detect macrolide antibiotic resistant mutant bacteria at low cost, accurately, and in a short time. In particular, by using a DNA polymerase that recognizes a primer having a difference of one base at the 3' end from the primer for detecting drug-resistant Mycoplasma pneumoniae used in the present invention and in which the amplification efficiency is significantly reduced in the presence of a mismatch pair, it is possible to distinguish the resistance gene mutation of Mycoplasma pneumoniae to macrolide antibiotics, and to determine the presence or absence of Mycoplasma pneumoniae mutant bacteria resistant to macrolide antibiotics. In addition, by using a DNA polymerase that recognizes a primer having a difference of one base at the 3' end from the primer for detecting drug-resistant Bordetella pertussis bacteria and in which the amplification efficiency is significantly reduced in the presence of a mismatch pair, it is possible to determine the presence or absence of Bordetella pertussis mutant bacteria resistant to macrolide antibiotics.

1 shows the results of PCR using the base sequence including the mutation of 23S rRNA, primers for detecting drug-resistant Mycoplasma pneumoniae bacteria, and HiDi DNA polymerase (registered trademark). The amplified PCR product was electrophoresed on a 2% agarose gel at 135 V for 20 minutes, and a specific amplification product was confirmed at 250 bp. (Example 1) 2 shows the results of PCR using the base sequence including the mutation of 23S rRNA, primers for detecting drug-resistant Bordetella pertussis bacteria, and HiDi DNA polymerase (registered trademark). The amplified PCR product was electrophoresed on a 2% agarose gel at 100 V for 30 minutes, and a specific amplification product was confirmed at 0.33 kbp. (Example 2)

Below, we will explain in detail the embodiments of the present invention, but these embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiments described below are also included in the scope of the present invention.

The present invention provides a method for detecting a mutant bacterium resistant to a macrolide antibiotic, which comprises detecting a point mutation of two adenines to which a macrolide antibiotic binds in the V region of the 23S rRNA domain of the mutant bacterium resistant to a macrolide antibiotic by PCR amplification using a DNA polymerase that recognizes a primer having a one-base difference at the 3' end and in which the amplification efficiency is significantly reduced in the presence of a mismatch pair.
In one embodiment, the present invention provides a method for detecting drug-resistant Mycoplasma pneumoniae bacteria, comprising detecting point mutations of adenines at positions 2063 and 2064 in the V region of the 23S rRNA domain of Mycoplasma pneumoniae (type strain M129; NCBI Reference Sequence: NC_000912.1) using HiDi DNA Polymerase (registered trademark).
In another embodiment, the present invention provides a method for detecting drug-resistant bacteria of Bordetella pertussis, comprising detecting point mutations of adenines at positions 2037 and 2038 in the V region of 23S rRNA domain of Bordetella pertussis (type strain Tohama I; NCBI Reference Sequence: NC_002929.2) using HiDi DNA Polymerase (registered trademark).
In the present specification, in the two adjacent adenines (e.g., positions 2058 or 2059 in E. coli, positions 2063 and 2064 in Mycoplasma pneumoniae, and positions 2037 and 2038 in Bordetella pertussis) in domain V of 23S rRNA to which macrolide antibiotics bind, for example, a point mutation in which adenine (A) at position 2058 in E. coli is replaced with guanine (G) is referred to as GA, a point mutation in which adenine (A) at position 2059 in E. coli is replaced with guanine (G) is referred to as AG, and when adenines (A) at positions 2058 and 2059 in E. coli are unchanged, they are also referred to as AA.

DNA polymerase extends a daughter DNA molecule in the 5' to 3' direction from the 3' end of the primer. The replicative fidelity of a DNA polymerase refers to the precision of polymerization and the ability of the DNA polymerase to distinguish between correct and incorrect nucleotides when synthesizing DNA complementary to the template. The higher the replicative fidelity of a DNA polymerase, the fewer nucleotides the DNA polymerase will misincorporate into the growing strand during DNA synthesis.

DNA polymerases with increased replication fidelity include, for example, Phusion DNA polymerase and Pyrococcus furiosus (Pfu) DNA polymerase.

DNA polymerases that recognize primers with a single base difference at the 3' end and in which the amplification rate drops significantly if there is no perfect match include HiDi DNA Polymerase (registered trademark).

A DNA polymerase that recognizes a primer with a single base difference at the 3' end and in which the amplification rate drops significantly if there is no perfect match is a DNA polymerase that recognizes a guanine (G)/cytosine (C) or adenine (A)/thymine (T) base pair with a single base at the 3' end of the primer and a single base at the corresponding position of the template DNA when the primer forms a primer/template complex with the template DNA, and does not form a G/C or A/T base pair, meaning that the amplification rate by the DNA polymerase drops when a mismatch pair occurs.

HiDi DNA Polymerase (registered trademark) (High Discrimination Taq DNA Polymerase) is a highly selective DNA polymerase variant genetically engineered based on the Klenow fragment of Taq DNA polymerase (M. Drum et al., PLoS One. 2014 May6;9(5):e96640). It can be selected for assays requiring high discrimination, for example, in allele-specific PCR or methylation-specific PCR. While many DNA polymerases tolerate mismatched primer/template complexes, HiDi DNA Polymerase (registered trademark) efficiently discriminates between them and generates specific amplification products only in the case of perfectly matched primer pairs. HiDi DNA Polymerase (registered trademark) recognizes primers with a single base difference at the 3' end, and the amplification rate is significantly reduced if there is no perfect match.

In a preferred embodiment, the DNA polymerase of the present invention is HiDi DNA polymerase (registered trademark).

As used herein, a "primer" refers to a single-stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a DNA molecule. Primers are preferably 15-40 bases in length, more preferably 20-35 bases in length. For example, the 3' end of the primer provides a free 3'-OH group to which additional nucleotides can be attached by a DNA polymerase establishing a 3'→5' phosphodiester bond.

In this specification, a "primer for detecting drug-resistant Mycoplasma pneumoniae bacteria" is a primer used to detect a nucleic acid molecule encoded by Mycoplasma pneumoniae bacteria that are resistant to macrolide antibiotics, and is a primer used to detect point mutations of adenines at positions 2063 and 2064 in the 23S rRNA domain V region of Mycoplasma pneumoniae (standard strain M129; NCBI Reference Sequence: NC_000912.1). One embodiment of the primers for detecting drug-resistant Mycoplasma pneumoniae bacteria is preferably a set including a 20-35 base-long AG detection primer having a sequence of 5'-GTAAAGCTTCACGGGGTCTC -3' on the 3' side, a 20-35 base-long AA detection primer having a sequence of 5'-TAAGCTTCACGGGGTCTTT -3' on the 3' side, and a 20-35 base-long GA detection primer having a sequence of 5'-TAAGCTTCACGGGGTCTTC -3' on the 3' side and a forward primer having a 20-40 base-long primer sequence having a sequence of 5'-CTGCCCAGTGCTGGAAGGTT-3' on the 5' side. More preferably, the primers are a set including a reverse primer having a sequence of any one of SEQ ID NOs: 1 to 3 and a forward primer having a sequence of SEQ ID NO: 4.

In this specification, the "primers for detecting drug-resistant Bordetella pertussis bacteria" are primers used to detect nucleic acid molecules encoded by drug-resistant bacteria of Bordetella pertussis against macrolide antibiotics, and are primers used to detect adenine point mutations at positions 2037 and 2038 in the 23S rRNA domain V region of Bordetella pertussis (standard strain Tohama I; NCBI Reference Sequence: NC_002929.2). One embodiment of the primers for detecting drug-resistant Bordetella pertussis bacteria is preferably a set including a 20-35 base-long reverse primer including any one of SEQ ID NOs: 5 to 11 for AA detection, any one of SEQ ID NOs: 12 to 18 for AG detection, and any one of SEQ ID NOs: 19 to 25 for GA detection on the 3' side, and a 20-40 base-long forward primer including any one of SEQ ID NOs: 26 to 29 for primer sequence on the 5' side. More preferably, the set includes a primer having a base sequence of any one of SEQ ID NOs: 5 to 11, a primer having a base sequence of any one of SEQ ID NOs: 12 to 18, and a reverse primer having a base sequence of any one of SEQ ID NOs: 19 to 25, and a forward primer having a base sequence of any one of SEQ ID NOs: 26 to 29. Even more preferably, the set includes a primer having a base sequence of any one of SEQ ID NOs: 5 to 11, a primer having a base sequence of any one of SEQ ID NOs: 12, 13, 15, and 16, and a reverse primer having a base sequence of any one of SEQ ID NOs: 20, 22, and 23, and a forward primer having a base sequence of any one of SEQ ID NOs: 27 to 29. Most preferably, the set includes a primer having a base sequence of SEQ ID NO: 6, a primer having a base sequence of SEQ ID NO: 13, and a reverse primer having a base sequence of SEQ ID NO: 20, and a forward primer having a base sequence of SEQ ID NO: 29.

An oligonucleotide is a synthetic or natural molecule that contains a covalent sequence of nucleotides linked by a phosphodiester bond between the 3' position of the pentose sugar of one nucleotide and the 5' position of the pentose sugar of an adjacent nucleotide. Oligonucleotides can be used as primers and probes.

As used herein, "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides include deoxyribonucleoside triphosphates (e.g., dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof). As used herein, nucleotides also refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. In accordance with the present invention, a "nucleotide" can be unlabeled or detectably labeled by well-known techniques. Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

Mycoplasma pneumoniae is classified as a bacterium because it does not require host cells for self-proliferation and some antibiotics are effective against it. However, Mycoplasma pneumoniae does not have a cell wall, which is a characteristic of bacteria, and therefore is resistant to β-lactam antibiotics (penicillin, cephalosporin, etc.), which are used as the first choice for treating bacterial infections and have the effect of damaging the bacterial cell wall and killing the bacteria, and macrolide antibiotics (erythromycin (EM), clarithromycin (CAM), azithromycin (AZM), etc.), which are protein synthesis inhibitors, are considered effective against it.

In this specification, "macrolide antibiotic-resistant mutant bacteria" refers to bacteria that have acquired resistance to macrolide antibiotics due to gene mutation. Macrolide antibiotic-resistant mutant bacteria include Mycoplasma pneumoniae , Bordetella pertussis , Helicobacter pylori , Campylobacter jejuni , Chlamydia trachomatis , Mycobacterium avium , and Mycobacterium intracellulare, which are drug-resistant bacteria against macrolide antibiotics. It has been revealed that the occurrence of macrolide antibiotic-resistant mutant bacteria is closely correlated with 23S rRNA gene mutation. In this specification, "Mycoplasma pneumoniae drug-resistant bacteria" refers to Mycoplasma pneumoniae that has drug resistance against macrolide antibiotics.

The two adjacent adenines in domain V of 23S rRNA to which macrolide antibiotics bind are at positions 2058 and 2059 in Escherichia coli, which is the most widely studied bacterium, but are at positions 2063 and 2064 in Mycoplasma pneumoniae (type strain M129; NCBI Reference Sequence: NC_000912.1), 2037 and 2038 in Bordetella pertussis (type strain Tohama I; NCBI Reference Sequence: NC_002929.2), 2146 and 2147 in Helicobacter pylori (type strain Helicobacter pylori 26695; NCBI Reference Sequence: NC_000915.1) (some sources consider them to be positions 2142 and 2143 based on the sequence of Accession number U27270), and Campylobacter jejuni (type strain Campylobacter jejuni subsp. jejuni The positions 2074 and 2075 in Chlamydia trachomatis (type strain Chlamydia trachomatis D/UW-3/CX;NCBI Reference Sequence:NC_000117.1), 2038 and 2039 in Mycobacterium avium (type strain Mycobacterium avium 104;NCBI Reference Sequence: NC_008595.1), and 2266 and 2267 in Mycobacterium intracellulare (type strain Mycobacterium intracellulare ATCC 13950;NCBI Reference Sequence:NC_016946.1) correspond to the positions of two adjacent adenines in domain V of 23S rRNA to which macrolide antibiotics bind. By changing the primer for detecting drug-resistant Mycoplasma pneumoniae bacteria or the primer for detecting drug-resistant Bordetella pertussis bacteria used in the present invention to one that corresponds to the positions of two adjacent adenines in domain V of 23S rRNA of the above-mentioned macrolide antibiotic-resistant mutant bacteria, it becomes possible to detect the above-mentioned macrolide antibiotic-resistant mutant bacteria.

That is, according to the present invention, in addition to Mycoplasma pneumoniae and Bordetella pertussis , the presence or absence of macrolide antibiotic-resistant mutant bacteria can be detected by examining 23S rRNA gene mutations in Helicobacter pylori , Campylobacter jejuni , Chlamydia trachomatis , Mycobacterium avium , and Mycobacterium intracellulare, for which macrolide antibiotics are positioned as the first or main drugs of choice.

As used herein, "amplification" refers to any in vitro method for increasing the number of copies of a nucleotide sequence using a DNA polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer, thereby forming a new DNA molecule complementary to the DNA template. The formed DNA molecule and its template can be used as a template for synthesizing additional DNA molecules. As used herein, an amplification reaction consists of many rounds of DNA replication. DNA amplification reactions include, for example, polymerase chain reaction (PCR). One PCR may consist of 20 to 100 "cycles" of denaturation and synthesis of a DNA molecule, usually including thermal denaturation of the template DNA molecule, annealing of the primers, and extension of the primers. Reaction conditions for PCR include the properties of the DNA polymerase, the type of PCR buffer, the complexity of the template DNA, and the melting temperature (Tm) of the primers.

PCR is usually performed using a thermal cycler. In this specification, the thermal cycler conditions refer to the PCR temperature and time settings used in the thermal cycler. In one embodiment of Touchdown PCR, which reduces non-specific PCR amplification products and promotes specific amplification, the thermal cycler conditions are (1) 98°C, 1 minute, 1 cycle, followed by (2) 98°C, 10 seconds, (3) 72°C to 56°C, 15 seconds, and (4) 72°C, 30 seconds, for a total of 36 cycles consisting of (2) to (4), followed by (5) 72°C, 1 minute, 1 cycle, where the step (3) is always performed for 15 seconds, but the temperature in the first cycle starts at 72°C, is lowered by 1°C every cycle to 57°C in the 16th cycle, and the 17th to 36th cycles are performed at 56°C. The thermal cycler conditions can be set under conditions known to those skilled in the art, but are not limited to these. For example, when Touchdown PCR is not performed, the temperature in the above step (3) can be constant at a temperature used in normal PCR in the range of 40° C. to 70° C., preferably in the range of 50° C. to 70° C., and most preferably at 60° C. In this step, the lower the temperature, the more likely unintended sequences are to be amplified, and the higher the temperature, the lower the annealing efficiency, i.e., the lower the amplification efficiency.

PCR is a process that uses DNA polymerase and deoxyribonucleoside triphosphates to amplify a target DNA template. In such PCR, two primers (one primer is complementary to the 3' end of the first strand of the DNA molecule to be amplified and the other primer is complementary to the 3' end of the second strand of the DNA molecule to be amplified) are hybridized to their respective DNA strands. After hybridization, DNA polymerase, in the presence of deoxyribonucleoside triphosphates, allows the synthesis of a third DNA molecule complementary to the first strand and a fourth DNA molecule complementary to the second strand of the DNA molecule to be amplified. This synthesis results in two double-stranded DNA molecules. Such double-stranded DNA molecules can then be used as DNA templates for the synthesis of additional DNA molecules by providing DNA polymerase, primers, and deoxyribonucleoside triphosphates.

The test nucleic acid in the present invention may be, for example, single-stranded or double-stranded. In the case of a double-stranded nucleic acid, it is preferable to include a step of dissociating the double stranded nucleic acid into a single strand by heating, for example, in order to hybridize the test nucleic acid with the probe to form a hybrid.

"Detecting an amplification reaction" includes detecting the amplification product by PCR by electrophoresis in a gel and staining the nucleic acid in the gel to detect it as a band corresponding to the base length of the nucleic acid, and detecting a fluorescent probe by real-time PCR.
Known methods for detecting fluorescence in real-time PCR include the intercalator method, which uses a fluorescent dye that binds to double-stranded DNA, the hydrolysis probe method, which uses a fluorescent probe that specifically binds to a gene product, and the HybProbe method.
The intercalator method is highly versatile because a fluorescent dye that binds to double-stranded DNA can be used for any sequence.
The hydrolysis probe method uses a hydrolysis probe that contains a base sequence complementary to the PCR amplification product to be detected. The hydrolysis probe is a probe in which a fluorescent dye is labeled at the 5' end and a quencher is labeled at the 3' end of an oligonucleotide specific to a target nucleic acid. When the probe is hydrolyzed by the 5'-3' exonuclease activity of the PCR enzyme, a fluorescent signal is emitted, and by detecting this, trace amounts of nucleic acid can be detected and quantified.
In the HybProbe method, two probes, one of which is labeled with a fluorescent dye at its 3' end and contains a base sequence complementary to the PCR amplification product to be detected, and the other is labeled with LightCycler (registered trademark) Red at its 5' end and has a phosphorylated 3' end, are hybridized in close proximity to each other, causing fluorescence resonance energy transfer (FRET) between the fluorescent substances, emitting a fluorescent signal, which can be detected to detect or quantitate nucleic acids.
The probe can be modified in design so that the primer for detecting drug-resistant Mycoplasma pneumoniae bacteria of the present invention can detect mutations in two adjacent adenines in domain V of 23S rRNA to which macrolide antibiotics bind, and the presence or absence of macrolide antibiotic-resistant mutant bacteria can be detected by examining 23S rRNA gene mutations using a fluorescent detection method for real-time PCR.

The type of test nucleic acid is not particularly limited, but examples thereof include DNA, total RNA, mRNA, and other RNA. Examples of the test nucleic acid include nucleic acids contained in samples such as Mycoplasma pneumoniae culture samples, catheter washing fluid, and biological samples. These samples may be used as is, or may be diluted with an appropriate solution. Examples of appropriate solutions include water, physiological saline, buffer solutions, alkaline aqueous solutions, acidic aqueous solutions, nucleic acid extraction reagents, surfactants, and organic solvents.

Biological samples are not particularly limited, but examples include pus, cerebrospinal fluid, pleural effusion, nasal swabs, pharyngeal swabs, sputum, bronchial lavage fluid, tissue slices, blood (whole blood), etc. There are no limitations on the method of collecting the sample, the method of preparing nucleic acids such as DNA and RNA, etc., and conventionally known methods can be used.

According to the present invention, three types of reverse primers (primers corresponding to wild-type strain, A2063G, and A2064G: SEQ ID NOs: 1 to 3) in which adenines at positions 2063 and 2064 of the 23S rRNA sequence of Mycoplasma pneumoniae are located at the 3' end are prepared, and a PCR method is performed on test nucleic acids obtained from subjects using a DNA polymerase containing HiDi DNA Polymerase (registered trademark) that recognizes primers having a difference of one base at the 3' end and in which the amplification efficiency is significantly reduced in the case of a perfect match. This makes it possible to distinguish, under a single PCR condition, between A2063G (a mutation in which adenine (A) at position 2063 is replaced with guanine (G)) and A2064G (a mutation in which adenine (A) at position 2064 is replaced with guanine (G)), which are the most common point mutations.

According to the present invention, three types of reverse primers (a primer consisting of any one of the base sequences of SEQ ID NOs: 5 to 11, a primer consisting of any one of the base sequences of SEQ ID NOs: 12 to 18, and a primer consisting of any one of the base sequences of SEQ ID NOs: 19 to 25) in which the adenines at positions 2037 and 2038 of the 23S rRNA sequence of Bordetella pertussis are located at the 3' end are prepared, and HiDi By carrying out PCR on test nucleic acids obtained from subjects using a DNA polymerase (registered trademark) that recognizes a primer having a one-base difference at the 3' end and in the case of a mismatch, the amplification efficiency of which is significantly reduced, it is possible to distinguish, under a single PCR condition, among the most common point mutations, A2037G (a mutation in which adenine (A) at position 2037 is replaced with guanine (G)), A2038G (a mutation in which adenine (A) at position 2038 is replaced with guanine (G)), and no mutation in which positions 2037 and 2038 are adenine (A) and adenine (A), respectively.

In one embodiment, the present invention provides a 23S rRNA gene detection kit for macrolide antibiotic-resistant mutant bacteria, particularly for Mycoplasma pneumoniae or Bordetella pertussis . The macrolide antibiotic-resistant mutant bacteria detection kit for Mycoplasma pneumoniae of the present invention includes a primer set including a reverse primer having a base sequence shown in any one of SEQ ID NOs: 1 to 3 and a forward primer having a base sequence shown in SEQ ID NO: 4, and can be used to detect drug-resistant bacteria of Mycoplasma pneumoniae . The macrolide antibiotic-resistant mutant bacteria detection kit for Bordetella pertussis of the present invention includes a set including a reverse primer of a primer having a base sequence shown in any one of SEQ ID NOs: 5 to 11, a primer having a base sequence shown in any one of SEQ ID NOs: 12 to 18, and a primer having a base sequence shown in any one of SEQ ID NOs: 19 to 25, and a forward primer having a base sequence shown in any one of SEQ ID NOs: 26 to 29, and can be used to detect drug-resistant bacteria of Bordetella pertussis . The kit of the present invention preferably contains appropriate reagents necessary for a nucleic acid amplification reaction and/or a nucleic acid amplification product detection reaction.

The present invention will be described in detail below with reference to examples, but the technical scope of the present invention should not be interpreted as being limited by the description of the following examples. Those skilled in the art can carry out many modifications and alterations based on the description in this specification without departing from the technical scope of the present invention. Furthermore, unless otherwise specified, the following examples can be carried out according to standard genetic engineering and molecular biology techniques known to those skilled in the art, such as those described in Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F.M.etal., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1995, etc. Furthermore, when using a kit, follow the instructions that come with the kit. The contents of the documents cited in this specification constitute the disclosure and part of the contents of this specification.

(Example 1) Detection of mutants of Mycoplasma pneumoniae resistant to macrolide antibiotics)
Using the throat swab of the subject, crude extraction of the test nucleic acid was performed by a general method. The specimen in the transport medium was used. 300 μl of the suspended specimen-containing medium was centrifuged at 4°C, 20,000 g, 30 minutes, and the sediment was suspended in Lysis Buffer containing Proteinase K, and incubated in a thermal cycler at 55°C, 60 minutes, 100°C, and 10 minutes, 4°C. In order to detect A2063G, in which the base adenine (A) at position 2063 of the wild type 23S rRNA of Mycoplasma pneumoniae is mutated to the base guanine (G), and A2064G, in which the base adenine (A) at position 2064 is mutated to the base guanine (G), from the test nucleic acid,
PCR reaction was carried out under optimized conditions using one forward primer (1838-CTGCCCAGTGCTGGAAGGTTAAAGAAGGAGGTTAG-1872 (SEQ ID NO: 4)) and three reverse primers (reverse primer (1): GCTACAGTAAGTTCCACGGGGTCTC (SEQ ID NO: 1), reverse primer (2): TTAAGCTACAGTAAAGCTTCACGGGGTCTTT (SEQ ID NO: 2), reverse primer (3): TTAAGCTACAGTAAAGCTTCACGGGGTCTTC (SEQ ID NO: 3)) and HiDi DNA polymerase (registered trademark) (Funakoshi). That is, a touchdown PCR was carried out under the following conditions using a thermal cycler to reduce non-specific PCR amplification products and promote specific amplification. (1) 98°C, 1 minute, 1 cycle, (2) 98°C, 10 seconds, (3) 72°C to 56°C, 15 seconds, (4) 72°C, 30 seconds, (2) to (4) were performed for a total of 36 cycles, and then (5) 72°C, 1 minute, 1 cycle was performed to end the experiment. The annealing step (3) was always performed for 15 seconds, but the temperature was changed or maintained depending on the cycle. That is, the temperature of the first cycle started at 72°C, and was lowered by 1°C for each cycle, from 71°C for the second cycle to 70°C for the third cycle, and so on, until the 16th cycle, 57°C, was similarly lowered by 1°C for each cycle. The 17th cycle was 56°C, and the 18th to 36th cycles were also performed at 56°C.
The amplified PCR products were electrophoresed on a 2% agarose gel at 135 V for 20 minutes, and the lanes in which a specific amplification product (about 250 bp) was confirmed were determined to be positive (FIG. 1).

(Example 2) Detection of mutants of Bordetella pertussis resistant to macrolide antibiotics)
A Bordetella pertussis strain isolated from a clinical specimen was subjected to nucleic acid extraction using QIAGEN's QIAampDNA mini kit. The nucleic acid was purified, and the upstream and downstream regions of the two bases (positions 2037 and 2038) that are mutated in the base sequence of the 23S rRNA sequence of Bordetella pertussis were amplified by PCR separately. These PCR amplified products were purified using Takara Bio's Gel and PCR clean up kit, and then "bridged" and joined together with a primer corresponding to 70 bases from 2003 to 2072. The primers used for purification are as follows (primer synthesis was outsourced to Eurofins Genomics, Inc.):
Forward (BpSC-F-T7)
GATAATACGACTCACTATAGGGCATGAAGGGGTCGCAGAGAATCGG (SEQ ID NO: 30)
Reverse (BpSC-A-Rev)
CCGTCTAGCCGCGGGTAGATTGCATCATCA (SEQ ID NO:31)

Forward (BpSC-B-Fwd)
AGACCCCATGAACCTTTACTGTAGCTTTGC (SEQ ID NO: 32)
Reverse (BpSC-R-T3)
5'-GAAATTAACCCTCACTAAAGGGCCCAGCTCACGTACCTCTTTAAATGGCG-3' (SEQ ID NO: 33)

And, during the above bridge,
For AA detection, a 70-base primer (BpSC-AA) (TTTGTGATGATGCAATCTACCCGCGGCTAGACGGAAAGACCCCATGAACCTTTACTGTAGCTTTGCATTG: SEQ ID NO: 34) in which both positions 2037 and 2038 are adenines (A) was used.
For AG detection, a 70-base primer (BpSC-AG) (TTTGTGATGATGCAATCTACCCGCGGCTAGACGGAGAGACCCCATGAACCTTTACTGTAGCTTTGCATTG: SEQ ID NO: 35) was used in which positions 2037 and 2038 were adenine (A) and guanine (G), respectively.
For GA detection, a 70-base primer (BpSC-GA) (TTTGTGATGATGCAATCTACCCGCGGCTAGACGGGAAGACCCCATGAACCTTTACTGTAGCTTTGCATTG: SEQ ID NO: 36) was used in which positions 2037 and 2038 were guanine (G) and adenine (A), respectively.
The PCR product thus prepared was purified again to prepare a nucleic acid sample at 10 ³ copies/μL, which was used as the test nucleic acid.

In order to detect adenine mutations at positions 2037 and 2038 of 23S rRNA of Bordetella pertussis from a test nucleic acid, a PCR reaction was performed under optimized conditions using the following detection primers and HiDi DNA polymerase (registered trademark) (Funakoshi).

The primers for detecting AA, AG, and GA of Bordetella pertussis (primer synthesis was outsourced to Eurofins Genomics K.K.) are as follows.

Forward primer (4 types)
BpFwd11 1730- CATGAAGGGGTCGCAGAGAATCG -1752 (SEQ ID NO: 26)
BpFwd12 1731-ATGAAGGGGTCGCAGAGAATCGG-1753 (SEQ ID NO: 27)
BpFwd13 1730- CATGAAGGGGTCGCAGAGAATCGG -1753 (SEQ ID NO: 28)
BpFwd14 1729- GCATGAAGGGGTCGCAGAGAATCGG -1753 (SEQ ID NO: 29)

Reverse primer: Primer for detecting AA (adenine (A) at positions 2037 and 2038)
BpAA12 2067 5'-CAAAGCTACAGTAAAGGTTCATGGGGTCTTT-3' 2037 (SEQ ID NO:5)
BpAA10 2065 AAGCTACAGTAAAGGTTCATGGGGTCTTT (SEQ ID NO: 6)
BpAA08 2063 GCTACAGTAAAGGTTCATGGGGTCTTT (SEQ ID NO: 7)
BpAA06 2061 TACAGTAAAGGTTCATGGGGTCTTT (SEQ ID NO: 8)
BpAA04 2059 CAGTAAAGGTTCATGGGGTCTTT (SEQ ID NO: 9)
BpAA02 2057 GTAAAGGTTCATGGGGTCTTT (SEQ ID NO: 10)
BpAA00 2055 AAAGGTTCATGGGGTCTTT (SEQ ID NO: 11)

Primer for detecting AG (mutation at position 2038)
BpAG12 2067 CAAAGCTACAGTAAAGGTTCATGGGGTCTC 2038 (SEQ ID NO: 12)
BpAG10 2065 AAGCTACAGTAAAGGTTCATGGGGTCTC (SEQ ID NO: 13)
BpAG08 2063 GCTACAGTAAAGGTTCATGGGGTCTC (SEQ ID NO: 14)
BpAG06 2061 TACAGTAAAGGTTCATGGGGTCTC (SEQ ID NO: 15)
BpAG04 2059 CAGTAAAGGTTCATGGGGTCTC (SEQ ID NO: 16)
BpAG02 2057 GTAAAGGTTCATGGGGTCTC (SEQ ID NO: 17)
BpAG00 2055 AAAGGTTCATGGGGTCTC (SEQ ID NO: 18)

Primer for detecting GA (mutation at position 2037)
BpGA12 2067 CAAAGCTACAGTAAAGGTTCATGGGGTCTTC 2037 (SEQ ID NO: 19)
BpGA10 2065 AAGCTACAGTAAAGGTTCATGGGGTCTTC (SEQ ID NO: 20)
BpGA08 2063 GCTACAGTAAAGGTTCATGGGGTCTTC (SEQ ID NO: 21)
BpGA06 2061 TACAGTAAAGGTTCATGGGGTCTTC (SEQ ID NO: 22)
BpGA04 2059 CAGTAAAGGTTCATGGGGTCTTC (SEQ ID NO: 23)
BpGA02 2057 GTAAAGGTTCATGGGGTCTTC (SEQ ID NO: 24)
BpGA00 2055 AAAGGTTCATGGGGTCTTC (SEQ ID NO: 25)

That is, Touchdown PCR was performed using a thermal cycler under the following conditions to reduce nonspecific PCR amplification products and promote specific amplification. (1) 98°C, 1 minute, 1 cycle, (2) 98°C, 10 seconds, (3) 72°C to 56°C, 15 seconds, (4) 72°C, 30 seconds, a total of 36 cycles consisting of (2) to (4), and then (5) 72°C, 1 minute, 1 cycle was performed to end. The annealing step (3) is always performed for 15 seconds, but the temperature was changed or maintained depending on the cycle. That is, the temperature of the first cycle started at 72°C, the second cycle was 71°C, the third cycle was 70°C, and so on, and was similarly decreased by 1°C for each cycle until the 16th cycle was 57°C. The 17th cycle was 56°C, and the 18th to 36th cycles were also performed at 56°C.
The amplified PCR products were electrophoresed on a 2% agarose gel at 100 V for 30 minutes to confirm the presence or absence of a specific amplification product (0.33 kbp) (FIG. 2).
The legend to FIG. 2 is as follows:
Lane M indicates a 100 bp ladder marker.
Lanes 1 to 3 show negative controls (NTC, no template control).
Lanes 1, 4, 5, and 6 show the results of PCR amplification using a combination of a forward primer (BpFwd14: GCATGAAGGGGTCGCAGAGAATCGG (sequence number 29)) and a reverse primer (BpAA10: AAGCTACAGTAAAAGGTTCATGGGGTCTTT (sequence number 6)) and a target NTC/AA/AG/GA sample as a template; an amplification product (0.33 kbp) was confirmed only for the AA sequence.
Lanes 2, 7, 8, and 9 show the results of PCR amplification using a combination of a forward primer (BpFwd14: GCATGAAGGGGTCGCAGAGAATCGG (sequence number 29)) and a reverse primer (BpAG10: AAGCTACAGTAAAGGTTCATGGGGTCTC (sequence number 13)) with the NTC/AA/AG/GA target sample as a template; an amplification product (0.33 kbp) was confirmed only for the AG sequence.
Lanes 3, 10, 11, and 12 show the results of PCR amplification using a combination of a forward primer (BpFwd14: GCATGAAGGGGTCGCAGAGAATCGG (sequence number 29)) and a reverse primer (BpGA10: AAGCTACAGTAAAAGGTTCATGGGGTCTTC (sequence number 20)) with the NTC/AA/AG/GA target sample as a template, and an amplification product (0.33 kbp) was confirmed only for the GA sequence.

Although the results are not shown here, in addition to the primer set used in Figure 2, the presence or absence of a specific amplification product (0.33 kbp) was confirmed by using a primer set consisting of a primer consisting of any one of the base sequences 5 to 11, a primer consisting of any one of the base sequences of SEQ ID NOs: 12, 13, 15, and 16, and a reverse primer consisting of a primer consisting of any one of the base sequences of SEQ ID NOs: 20, 22, and 23, and a forward primer consisting of any one of the base sequences of SEQ ID NOs: 27 to 29.

According to the present invention, it is possible to detect mutant bacteria resistant to macrolide antibiotics. In particular, by detecting point mutations of adenines at positions 2063 and 2064 in the 23S rRNA domain V region, which are important for drug resistance of Mycoplasma pneumoniae , and point mutations of adenines at positions 2037 and 2038 in the 23S rRNA domain V region, which are important for drug resistance of Bordetella pertussis , it is possible to detect drug-resistant bacteria of Mycoplasma pneumoniae and Bordetella pertussis against macrolide antibiotics accurately, quickly, and simply at low cost.

Claims (7)

A method for detecting a mutant bacterium resistant to a macrolide antibiotic, comprising detecting point mutations of two adenines to which a macrolide antibiotic binds in a 23S rRNA domain V region of the mutant bacterium resistant to a macrolide antibiotic by PCR amplification using a DNA polymerase that recognizes a primer having a difference of one base at the 3' end and in which the amplification efficiency is significantly reduced when a mismatch pair is present,
The macrolide antibiotic-resistant mutant bacteria are drug-resistant bacteria of Mycoplasma pneumoniae, and the two adenines to which the macrolide antibiotic binds in the 23S rRNA domain V region are located at positions 2063 and 2064 in the 23S rRNA domain V region of Mycoplasma pneumoniae (standard strain M129; NCBI Reference Sequence: NC_000912.1);
In PCR amplification, the reverse primer for detecting the adenine mutations at positions 2063 and 2064 of the 23S rRNA sequence of M. pneumoniae was:
- a primer having a length of 20 to 35 bases and containing the sequence shown in 5'-GTAAAGCTTCACGGGGTCTC-3' on the 3'side;
- a primer having a length of 20 to 35 bases and containing the sequence shown in 5'-TAAAGCTTCACGGGGTCTTT-3' on the 3' side, and
A primer of 20 to 35 bases in length containing the sequence shown in 5'-TAAAGCTTCACGGGGTCTTC-3' on the 3' side
How to use, or
the macrolide antibiotic-resistant mutant is a drug-resistant bacterium of Bordetella pertussis, and two adenines to which macrolide antibiotics bind in the 23S rRNA domain V region are located at positions 2037 and 2038 in the 23S rRNA domain V region of Bordetella pertussis (standard strain Tohama I; NCBI Reference Sequence: NC_002929.2);
In PCR amplification, the reverse primer for detecting the adenine mutations at positions 2037 and 2038 of the 23S rRNA sequence of Bordetella pertussis was:
A primer having a length of 20 to 35 bases and including a sequence shown in any one of SEQ ID NOs: 5 to 11 on the 3'side;
A primer having a length of 20 to 35 bases and including a sequence shown in any one of SEQ ID NOs: 12 to 18 on the 3'side; and
A primer having a length of 20 to 35 bases and containing a sequence shown in any one of SEQ ID NOs: 19 to 25 on the 3' side.
How to use . 2. The method of claim 1 , wherein the DNA polymerase is HiDi DNA polymerase. A reverse primer for detecting adenine mutations at positions 2063 and 2064 of the 23S rRNA sequence of Mycoplasma pneumoniae is a primer consisting of the sequence shown in 5'-GCTACAGTAAAGCTTCACGGGGTCTC-3' (SEQ ID NO: 1);
The method according to claim 1 , wherein the primer is a primer having the sequence shown in 5'-TTAAGCTACAGTAAAGCTTCACGGGGTCTTT-3' (sequence number 2), and the primer is a primer having the sequence shown in 5'-TTAAGCTACAGTAAAGCTTCACGGGGTCTTC-3' (sequence number 3). The method according to claim 1, wherein the reverse primers for detecting adenine mutations at positions 2037 and 2038 of the 23S rRNA sequence of Bordetella pertussis are a primer consisting of any one of the base sequences set forth in SEQ ID NOs: 5 to 11, a primer consisting of any one of the base sequences set forth in SEQ ID NOs: 12 to 18, and a primer consisting of any one of the base sequences set forth in SEQ ID NOs: 19 to 25 . The method according to any one of claims 1 to 4, wherein the thermal cycler conditions are (1) 1 cycle at 98°C for 1 minute, followed by (2) 98°C for 10 seconds, (3) 72°C to 56°C for 15 seconds, and (4) 72°C for 30 seconds, for a total of 36 cycles consisting of (2) to (4), and then (5) 1 cycle at 72°C for 1 minute, in which the step ( 3 ) is always performed for 15 seconds, but the temperature in the first cycle starts at 72°C and is lowered by 1°C every cycle until the 16th cycle at 57°C, and the 17th to 36th cycles are performed at 56°C. A kit for detecting macrolide antibiotic-resistant mutants of Mycoplasma pneumoniae, comprising:
A kit comprising a primer set including a reverse primer having the base sequence shown in any one of SEQ ID NOs: 1 to 3 and a forward primer having the base sequence shown in SEQ ID NO: 4. A kit for detecting a macrolide antibiotic-resistant mutant of Bordetella pertussis, comprising:
A kit comprising a primer set including a reverse primer of a primer consisting of a base sequence of any one of SEQ ID NOs: 5 to 11, a primer consisting of a base sequence of any one of SEQ ID NOs: 12 to 18, and a primer consisting of a base sequence of any one of SEQ ID NOs: 19 to 25, and a forward primer consisting of a base sequence of any one of SEQ ID NOs: 26 to 29.