Oligonucleotides for detecting Chlamydia pneumoniae

Abstract

To provide oligonucleotides for the detection of Chlamydia pneumoniae. [Solution] A probe for detecting the tyrp gene of Chlamydia pneumoniae is disclosed. The probe preferably has the following base sequence (A) or (B): (A) an oligonucleotide containing a base sequence S1 of at least 10 consecutive bases in a specific base sequence or a base sequence complementary thereto, or a base sequence S2 in which 1 to 3 bases are substituted, deleted, inserted, or added in base sequence S1. (B) only the 5' end or 3' end of the oligonucleotide of (A) is labeled.

Description

Technical Field

[0001] The present invention relates to oligonucleotides and the like for detecting Chlamydophila (Chlamydia) pneumoniae that may be contained in a sample. Further, the present invention relates to a method for detecting Chlamydophila pneumoniae that may be contained in a sample using the oligonucleotide, and reagents and kits for use in the method.

Background Art

[0002] Respiratory infections caused by Chlamydophila (Chlamydia) pneumoniae develop atypical pneumonia after a 3- to 4-week incubation period through droplet infection. When diagnosed with Chlamydia pneumoniae, reporting is required on a weekly basis, and rapid testing is important (Non-Patent Document 1).

[0003] Furthermore, differentiation from other respiratory infections represented by Mycoplasma pneumoniae is also important, and the contribution of rapid testing to early treatment is significant. As tests for Chlamydophila pneumoniae, antigen tests and nucleic acid amplification tests are being carried out, but there are concerns about insufficient sensitivity in antigen tests, and existing nucleic acid amplification tests have the problem of a long test time.

[0004] As a method for detecting nucleic acid amplification products, a melting curve analysis method is known. The melting curve analysis method can perform nucleic acid amplification and detection in separate steps, and can be measured relatively simply in about 30 minutes at the shortest. Furthermore, melting curve analysis using a fluorescently labeled nucleic acid probe has the advantage of being easily adaptable to gene tests using an automatic analyzer.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006] [Non-Patent Document 1] NIID (National Institute of Infectious Diseases): What is Chlamydia Pneumonia? IDWR2002, No. 7 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Patent Document 1 describes a method for distinguishing and detecting Chlamydia pneumoniae or Chlamydia psittacosis by targeting the ompA gene, which encodes the major outer membrane protein (MOMP) of these bacteria. The ompA gene is thought to exist as a single copy in the genomic DNA of these bacteria.

[0008] One object of the present invention is to provide a useful method for detecting Chlamydia pneumoniae that may be present in a sample. [Means for solving the problem]

[0009] The inventors conducted diligent research to achieve the above objectives and found that a method for detecting Chlamydia pneumoniae that targets the tyrp gene, which is present in multiple copies in the genomic DNA of Chlamydia pneumoniae, is useful. Based on this finding, further investigations led to the completion of the present invention.

[0010] The present invention encompasses embodiments described in the following sections. [Item 1] A probe for detecting the tyrp gene of Chlamydia pneumoniae. [Item 2] The probe described in Item 1, having the following base sequence (A) or (B): (A) Oligonucleotides containing an oligonucleotide sequence S1 of at least 10 consecutive bases in the base sequence 35-70 of Sequence ID No. 2 or Sequence ID No. 3 or its complementary base sequence, or a base sequence S2 in which 1-3 bases are substituted, deleted, inserted, or added to base sequence S1. (B) Only the 5' end or the 3' end of the oligonucleotide in (A) is labeled. [Item 3] The probe according to Item 2, wherein the length of the base sequence of (A) or (B) is 10 to 35 bases. [Clause 4] The probe according to Clause 2 or 3, wherein the base sequence of (A) or (B) includes the base sequence shown in any of Sequence IDs 8 to 13 or a complementary base sequence thereof. [Item 5] The probe according to any one of items 2 to 4, wherein the label is a fluorescent dye label. [Item 6] The probe according to any one of items 2 to 5, wherein the label is a fluorescent quenching dye that quenches when bound to a nucleic acid containing a base sequence that exhibits 90% or more identity with a base sequence complementary to the base sequence of the probe. [Item 7] The probe according to any one of items 2 to 6, wherein the label is a fluorescent quenching dye that is quenched by interaction with guanine. [Clause 8] The probe according to any one of Clauses 2 to 7, wherein the label is a fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives. [Clause 9] The probe according to any one of Clauses 2 to 8, wherein the label is labeled with at least one fluorescent quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 10] The probe according to any one of items 2 to 9, wherein the labeled terminal base is cytosine. [Item 11] A method for detecting Chlamydia pneumoniae that may be present in a sample using a probe described in any of Items 1 to 10. [Item 12] The following steps (1), (2), and (3): (1) A step of providing a sample that may contain Chlamydia pneumoniae, (2) A step of carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1), and The method according to claim 11, further comprising (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes. [Clause 13] The method according to Clause 12, wherein step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B. [Clause 14] The method according to Claim 13, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a variant thereof. [Item 15] The method according to any one of items 12 to 14, wherein the primer set for detecting Chlamydia pneumoniae used in the nucleic acid amplification reaction of step (2) comprises a first primer having a base sequence S3 of at least 15 consecutive bases in the base sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 bases are substituted, deleted, inserted or added in base sequence S3, and a second primer having a base sequence S5 of at least 15 consecutive bases in the base sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 bases are substituted, deleted, inserted or added in base sequence S5, wherein the second primer is complementary to the DNA elongation product of the first primer. [Clause 16] The method according to Claim 15, wherein the first primer has a nucleotide sequence shown in any of Sequence IDs 14 to 17 or a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 bases are substituted, deleted, inserted or added, and the second primer has a nucleotide sequence shown in any of Sequence IDs 18 to 22 or a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 bases are substituted, deleted, inserted or added. [Item 17] A method for distinguishing and detecting Chlamydia pneumoniae and other respiratory disease-causing bacteria that may be present in a sample, using a probe set comprising a probe described in any of Items 1 to 10 and a probe for detecting other respiratory disease-causing bacteria. [Item 18] The following steps (1), (2), and (3): (1) Providing a sample that may contain Chlamydia pneumoniae and other causative bacteria of respiratory diseases; (2) Performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1), a primer set for detecting Chlamydia pneumoniae, and a primer set for detecting other causative bacteria of respiratory diseases; and (3) Detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more of the probe sets, the method according to claim 17. [Claim 19] The probe for detecting other causative bacteria of respiratory diseases has a nucleotide sequence represented by SEQ ID NO: 25, The primer set for detecting other causative bacteria of respiratory diseases is a combination of a primer having a nucleotide sequence represented by SEQ ID NO: 23 and a primer having a nucleotide sequence represented by SEQ ID NO: 24, the method according to claim 18. [Claim 20] The detecting step in step (3) is performed by melting curve analysis, the method according to any one of claims 12 to 16. [Claim 21] A reagent or kit for detecting Chlamydia pneumoniae, comprising the probe according to any one of claims 1 to 10. [Claim 22] A reagent or kit for detecting Chlamydia pneumoniae, comprising the primer set according to claim 15 or 16. [Claim 23] A reagent or kit for distinguishing and detecting Chlamydia pneumoniae and other causative bacteria of respiratory diseases, comprising the probe according to any one of claims 1 to 10, a primer set for detecting Chlamydia pneumoniae, a probe for detecting other causative bacteria of respiratory diseases, and a primer set for detecting other causative bacteria of respiratory diseases. [Advantages of the Invention]

[0011] According to the present invention, useful means for detecting Chlamydia pneumoniae that may be contained in a sample can be provided. [Brief Description of the Drawings]

[0012] [Figure 1]It is a diagram showing a representative example of the results of Example 1 (a graph showing the detection results when melting curve analysis is performed using the probe represented by SEQ ID NO: 8). [Figure 2] It is a diagram showing a representative example of the results of Example 2 (a graph showing the detection results when melting curve analysis is performed using the probe represented by SEQ ID NO: 8). [Figure 3] It is a diagram showing a representative example of the results of Example 3 (a graph showing the detection results when performed with the combination of primers of SEQ ID NOs: 14 and 18). [Figure 4] It is a diagram showing a representative example of the results of Example 4 (a graph showing the detection results when melting curve analysis is performed using the probe represented by SEQ ID NO: 8). [Figure 5] It is a diagram showing a representative example of the results of Example 5 (a graph showing the detection results when melting curve analysis is performed using the probe represented by SEQ ID NO: 8). [Figure 6] It is a design drawing of primers and probes. [Figure 7] It is a design drawing of primers and probes.

MODE FOR CARRYING OUT THE INVENTION

[0013] Hereinafter, while showing embodiments of the present invention, the present invention will be described in more detail, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference in their entirety and are incorporated into the specification. In addition, "~" in this specification means "above and below". For example, if it is described as "X~Y" in the specification, it means "X or more and Y or less". "And / or" in this specification means any one or two or more possible combinations of the listed elements. "Comprising" in this specification includes the concepts of "consisting essentially of" and "consisting only of".

[0014] Furthermore, in this specification, nucleic acid primers may be simply referred to as primers, nucleic acid probes and labeled probes may be simply referred to as probes, and these are collectively referred to as oligonucleotides.

[0015] In one embodiment, the present invention provides a method for detecting Chlamydia pneumoniae using a probe (particularly a labeled probe) containing a nucleotide sequence targeting a specific gene region. In this method, by targeting the multicopy gene of Chlamydia pneumoniae, particularly the tyrp gene, it is possible to detect Chlamydia pneumoniae with high sensitivity in a short time, for example. In one embodiment, the present invention enables high sensitivity detection of Chlamydia pneumoniae, for example, in melting curve analysis, by designing a probe (particularly a labeled probe) targeting a specific nucleotide sequence region of the tyrp gene of Chlamydia pneumoniae. In this specification, SEQ ID NO: 1 is the full length of the tyrp gene of Chlamydia pneumoniae, and SEQ ID NOs: 2-7 are parts of the tyrp gene of Chlamydia pneumoniae. The present invention includes a probe (particularly a labeled probe) containing a specific nucleotide sequence targeting the nucleotide sequence region shown in SEQ ID NO: 2 or 3, and a primer containing a specific nucleotide sequence targeting the nucleotide sequence region shown in any of SEQ ID NOs: 4-7.

[0016] One embodiment of the present invention is a method for detecting Chlamydia pneumoniae that may be present in a sample. This method preferably uses a probe (particularly a labeled probe) having a specific base sequence, as described later. In a particular embodiment, by using one specific labeled probe in the reaction solution, Chlamydia pneumoniae that may be present in the sample can be detected with high sensitivity, for example, in PCR-melting curve analysis (including RT-PCR-melting curve analysis).

[0017] In a particular embodiment, the method of the present invention comprises at least the following steps (A) and (B): (A) A step of performing a nucleic acid amplification reaction using a nucleic acid primer set consisting of multiple nucleic acid primers containing a specific base sequence, using a specific region present in the base sequence of Chlamydia pneumoniae as a template, to produce one or more nucleic acid amplification products; and (B) A step of detecting one or more nucleic acid amplification products obtained in step (A) using one or more labeled probes of the present invention consisting of a specific base sequence. This includes a PCR-melting curve analysis method, in which a PCR reaction is performed in step (A) and a melting curve analysis method is performed in step (B). Steps (A) and (B) may be performed in the same reaction solution. Steps (A) and (B) may also be performed consecutively or simultaneously.

[0018] In a particular embodiment, a method for detecting Chlamydia pneumoniae that may be present in a sample comprises at least the following steps (1), (2), and (3): (1) A step of providing a sample that may contain Chlamydia pneumoniae, (2) A step of carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1), and (3) A step of detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes of the present invention. It is preferable that the method includes the following. It is preferable that step (2) be carried out by a PCR reaction and step (3) be carried out by melting curve analysis (PCR-melting curve analysis method). Steps (2) and (3) may be carried out in the same reaction solution. Alternatively, steps (2) and (3) may be carried out consecutively or simultaneously.

[0019] [Process (1)] In one embodiment, step (1) preferably involves preparing a sample that may contain Chlamydia pneumoniae (for example, biological samples such as animal and plant tissues, bodily fluids, excrement, and cells; environmental samples such as wipes from the walls, floors, equipment and fixtures of a facility, toilets, etc., or cleaning solutions used to clean them). The samples that can be used in the present invention are not particularly limited as long as they may contain Chlamydia pneumoniae. Examples include, but are not limited to, blood, blood culture medium, urine, pus, cerebrospinal fluid, pleural fluid, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, sputum, saliva, oral swabs, tissue sections, skin, vomit, feces, isolated culture colonies, catheter irrigation solutions, etc., taken from a subject suspected of being infected with Chlamydia pneumoniae. From the perspective that Chlamydia pneumoniae is a major causative agent of respiratory tract infections, in certain embodiments, it is preferable to use, for example, blood, blood culture medium, urine, pus, cerebrospinal fluid, pleural fluid, pharyngeal swab, nasal swab, nasopharyngeal swab, sputum, saliva, or oral swab as a sample, and it is even more preferable to use pharyngeal swab, nasal swab, nasopharyngeal swab, or sputum. When biological samples are to be measured, there are no particular limitations depending on the biological sample, but pretreatment such as dilution or suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, alkali treatment, organic solvent treatment, crushing treatment, grinding treatment, or nucleic acid extraction may or may not be performed.

[0020] The method of collecting and preparing the sample is not particularly limited, and known methods can be used depending on the type of sample, purpose, etc. In certain preferred embodiments, the sample does not have to be a sample from which DNA has been isolated or purified. For example, a sample collected from a living organism may be subjected to proteolytic denaturation treatment with a proteolytic enzyme (e.g., proteinase K) and / or heat treatment (e.g., 60-100°C for 1 second to 10 minutes) to degrade and remove the DNase (DNA-degrading enzyme) present in the sample, and this sample may be used as is.

[0021] The method of nucleic acid extraction is not particularly limited, but known methods can be used depending on the type of sample, purpose, etc. For nucleic acid extraction, kits sold by various manufacturers may be used, or automated nucleic acid extraction and purification equipment may be used.

[0022] [Process (2)] In one embodiment, step (2) is preferably a step of generating one or more nucleic acid amplification products by nucleic acid amplification (performing a nucleic acid amplification reaction using one or more nucleic acid primer sets). Nucleic acid amplification is a technique that amplifies several copies of a target nucleic acid to a level that can be visualized, i.e., hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in fields such as clinical diagnosis, food hygiene inspection, and environmental inspection. Examples of such nucleic acid amplification techniques include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, and TMA. These techniques are already established in the relevant field, and the method can be selected according to the purpose. The nucleic acid amplification technique is preferably PCR (including RT-PCR), but is not limited thereto.

[0023] (PCR reaction) PCR is a reaction primarily catalyzed by DNA polymerase. A PCR reaction typically involves a cycle consisting of three steps: (i) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (ii) annealing of primers to template single-stranded DNA, and (iii) extension of the primers using DNA polymerase. This cycle is repeated. Examples of DNA polymerases include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. In this invention, it is preferable to use a DNA polymerase belonging to Family B from the viewpoint of simplicity, speed, high sensitivity, and resistance to amplification inhibition by the sample. Furthermore, when step (3) is performed by melting curve analysis, it is preferable to use a DNA polymerase belonging to Family B that does not have 5'→3' exonuclease activity, from the viewpoint of using a fluorescent quenching probe.

[0024] The conditions for the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80-100°C for 0-300 seconds (e.g., 0.5-300 seconds), the second and subsequent (repeated) steps (i) may be performed at 80-100°C for 0.5-300 seconds, step (ii) may be performed at 35-80°C for 1-300 seconds, and step (iii) may be performed at 35-85°C for 1-300 seconds. It is preferable to repeat the cycle from steps (i) to (iii) 30-70 times. The temperature and time of the repeated cycles may be changed every 1-3 cycles.

[0025] (DNA polymerase) The DNA polymerase that can be used in step (2) is preferably a DNA polymerase belonging to Family B, but is not limited thereto. The DNA polymerase belonging to Family B is not particularly limited, but is preferably an archaeal DNA polymerase, and more preferably a DNA polymerase derived from bacteria of the genera Pyrococcus and Thermococcus. Also, suitable DNA polymerases include variants of archaeal DNA polymerases belonging to Family B that have not lost their DNA polymerase activity. Examples of variants include those in which 1 to 3 amino acids (e.g., 1 or 2, or 1) are deleted, substituted, inserted and / or added to the wild-type amino acid sequence, or variants that show 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more of amino acid sequence identity with the wild-type amino acid sequence. Specifically, DNA polymerase variants include, but are not limited to, variants aimed at enhancing polymerase activity, deficient in exonuclease activity, or adjusting substrate specificity.

[0026] DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp.GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as their mutants that have not lost DNA polymerase activity.

[0027] DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp.JDF-3, Thermococcus sp.9degrees North-7 (Thermococcus sp.9°N-7), Thermococcus siculi, and their mutants that have not lost DNA polymerase activity. Preferably, DNA polymerase derived from Thermococcus kodakaraensis and its mutants (for example, KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) can be used in the present invention particularly favorably from the viewpoint of excellent extensibility and thermal stability.

[0028] PCR enzymes using these DNA polymerases are commercially available, including Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0029] (KOD-derived DNA polymerase) In this specification, KOD-derived DNA polymerase (also called KOD DNA polymerase) refers to DNA polymerase derived from Thermococcus kodakaraensis and its variants (for example, KOD-derived DNA polymerases in which 3'→5' exonuclease activity is lost by substituting, deleting, inserting and / or adding 1 to 3 amino acids (e.g., 1 or 2, or 1) in the naturally occurring amino acid sequence). In one preferred embodiment, step (2) is to carry out a nucleic acid amplification reaction using such KOD-derived DNA polymerase. Compared to Taq DNA polymerase, which is a DNA polymerase belonging to Family A, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by inhibitors derived from the sample. In the present invention, using such KOD DNA polymerase is more preferable in that it is simple, rapid, and highly sensitive in detecting Chlamydia pneumoniae, as shown in the examples described later.

[0030] (Nucleic acid primer set) The nucleic acid primer set that can be used in step (2) is not particularly limited as long as it can amplify nucleic acid fragments derived from Chlamydia pneumoniae that can form a complex with the probe described later. From the viewpoint of obtaining more sensitive results, it is preferable that the nucleic acid primer set is a nucleic acid primer set that can amplify at least the base sequence shown in SEQ ID NO: 1, with Chlamydia pneumoniae as the amplification target.

[0031] In one embodiment, the nucleic acid primer set preferably includes a first primer having a base sequence S3 of at least 15 consecutive bases in the base sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or its complementary base sequence, or a base sequence S4 in which 1 to 3 bases are substituted, deleted, inserted, or added in base sequence S3, and a second primer having a base sequence S5 of at least 15 consecutive bases in the base sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary base sequence, or a base sequence S6 in which 1 to 3 bases are substituted, deleted, inserted, or added in base sequence S5.

[0032] The second primer is preferably complementary to the DNA elongation product of the first primer.

[0033] In a more preferred embodiment, the nucleic acid primer set comprises a first primer having a nucleotide sequence S31 represented by any of SEQ ID NOs: 14 to 17 or a complementary nucleotide sequence S31 thereto, or a nucleotide sequence S41 obtained by substituting, deleting, inserting, or adding 1 to 3 (e.g., 1 or 2, or 1) bases in nucleotide sequence S31, and a second primer having a nucleotide sequence S51 represented by any of SEQ ID NOs: 18 to 22 or a complementary nucleotide sequence S51 thereof, or a nucleotide sequence S61 obtained by substituting, deleting, inserting, or adding 1 to 3 (e.g., 1 or 2, or 1) bases in nucleotide sequence S51, wherein one primer is complementary to the DNA elongation product of the other primer. From the viewpoint of easily detecting pneumoniae with high sensitivity and a high fluorescence change rate even when detecting small amounts (e.g., about 1 to 20 copies), the nucleic acid primer set that can be used in the present invention is particularly preferable to be a nucleic acid primer set that includes a first primer having a base sequence S32 shown in SEQ ID NO: 14 or SEQ ID NO: 16 or its complementary base sequence S32, or a base sequence S42 obtained by substituting, deleting, inserting, or adding 1 to 3 bases (e.g., 1 or 2, or 1) in base sequence S32, and a second primer having a base sequence S62 shown in SEQ ID NO: 18, 19, or SEQ ID NO: 21 or its complementary base sequence S52, or a base sequence S62 obtained by substituting, deleting, inserting, or adding 1 to 3 bases (e.g., 1 or 2, or 1) in base sequence S52, wherein one primer is complementary to the DNA extension product of the other primer.

[0034] [Process (3)] The embodiments of step (3) are not particularly limited and can be carried out by any method known in the art. Chlamydia pneumoniae, the target of detection, can mutate like other infectious microorganisms. If a mismatch occurs between the base sequence of the primer or probe and the base sequence of the target Chlamydia pneumoniae variant, the binding affinity of the primer or probe to the target gene or the nucleic acid amplification product derived therefrom (target nucleic acid) may decrease. In particular, if there is a large mismatch between the target nucleic acid and the probe in real-time PCR, the probe may not be able to bind sufficiently to the target nucleic acid, which may cause the rise of the amplification curve to appear delayed or not rise at all, and such a situation can induce false negatives. In the case of melting curve analysis, since step (3) is performed after PCR is completed, even if there is a mismatch in the primer or probe, detection is possible if the nucleic acid amplification product is ultimately obtained, thus suppressing the effect of mismatches more than in real-time PCR. Therefore, in step (3), it is particularly preferable to detect the nucleic acid amplification product by melting curve analysis. Furthermore, by detecting nucleic acid amplification products using melting curve analysis, detection can be achieved in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less, from the start of the nucleic acid amplification reaction to the completion of the melting curve analysis).

[0035] In one embodiment, step (3) is steps (3-1) and (3-2): (3-1) A step of hybridizing one or more nucleic acid amplification products obtained in step (2) with one or more nucleic acid probes of the present invention to form a complex; and (3-2) Step to detect the composite of step (3-1) It is preferable to include the following. In the detection of Chlamydia pneumoniae, in order to obtain highly sensitive results, it is preferable to use a nucleic acid probe described later that can specifically react with the nucleic acid amplification product that can be produced in the nucleic acid amplification reaction of step (2) to form a complex. The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization between the nucleic acid amplification product and the nucleic acid probe. Such temperature conditions include, but are not limited to, a temperature at least 5°C lower, more preferably at least 10°C lower, than the Tm value of the nucleic acid probe.

[0036] (Nucleic acid probes) The nucleic acid probe of the present invention is not particularly limited as long as it can detect the tyrp gene of Chlamydia pneumoniae. In one embodiment, the nucleic acid probe of the present invention is preferably not a TaqMan probe, and it is preferably labeled only at either the 5' or 3' end. In a particular embodiment, the nucleic acid probe of the present invention preferably has the base sequence (A) or (B). When the nucleic acid probe of the present invention is a nucleic acid probe labeled with a fluorescent quenching dye that is quenched by interaction with guanine as described later, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0037] In one embodiment, the nucleic acid probe of the present invention can detect Chlamydia pneumoniae (including variants of Chlamydia pneumoniae) having a nucleotide sequence that shows 85% or more identity with any of SEQ ID NOs: 1 to 7 (for example, SEQ ID NO: 2 or SEQ ID NO: 3), and preferably a nucleic acid probe that can detect Chlamydia pneumoniae (including variants of Chlamydia pneumoniae) having a nucleotide sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0038] In certain embodiments, the nucleic acid probe of the present invention may be a probe containing a base sequence in which 1 to 3 bases are substituted, deleted, inserted, or added in base sequence A1 or B1. In this specification, "substitution" is used to include substitution of DNA bases with artificial nucleic acid bases such as LNA bases (e.g., T → LNA-T). The number of bases that can be substituted, deleted, inserted, or added is preferably 1 or 2. When a probe has such a substitution, deletion, insertion, or addition of bases, it is said that the probe contains mismatched bases, and it has been confirmed in the examples described later that probes containing such mismatched bases can also be suitably used in the present invention.

[0039] In this specification, "containing mismatched bases" (or simply "mismatched bases") means containing bases that are not complementary to the base sequence of the target nucleic acid (or, if the target nucleic acid becomes double-stranded after a nucleic acid amplification reaction, the base sequence of either single-stranded nucleic acid after the double-stranded nucleic acid dissociates). For example, if a cytosine base is present in the base sequence of the target nucleic acid, this means that the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., adenine bases, cytosine bases, thymine bases, and universal bases). For example, when detecting a mutable region of the target nucleic acid, mismatched bases (e.g., universal bases, mixed bases) can be selected at the probe position corresponding to the mutable base.

[0040] If the nucleic acid probe of the present invention contains a mismatched base, the position of the mismatched base is not particularly limited as long as it does not hinder the effects of the present invention. From the viewpoint of making it easier to detect nucleic acid amplification products more reliably, it is preferable that the mismatched base is not a terminal base of each probe. For example, the position of the mismatched base is preferably within 5 mers before or after the center of the total length n of the base sequence constituting the probe (if n is odd, (n+1) / 2; if n is even, n / 2), more preferably within 4 mers before or after, even more preferably within 3 mers before or after, even more preferably within 2 mers before or after, and particularly preferably within 1 mer before or after.

[0041] From the viewpoint of enabling more sensitive detection, the base sequence of (A) or (B) preferably includes the base sequence shown in any of SEQ ID NOs: 8 to 13 or a complementary base sequence thereof, more preferably includes the base sequence shown in any of SEQ ID NOs: 8, 9, and 12 or a complementary base sequence thereof, even more preferably includes the base sequence shown in any of SEQ ID NOs: 8 and 12 or a complementary base sequence thereof, and most preferably includes the base sequence shown in SEQ ID NO: 8 or a complementary base sequence thereof.

[0042] The length of the nucleic acid probe of the present invention is not particularly limited, and is, for example, 10 bases or more or 11 bases or more, and is usually 25 bases or less, preferably 23 bases or less, more preferably 22 bases or less, even more preferably 21 bases or less, and even more preferably 20 bases or less (for example, 19 bases or less, or 18 bases or less). The length of the nucleic acid probe of the present invention may be, for example, 10 to 35 bases, more preferably 16 to 22 bases, and even more preferably 16 to 21 bases. In certain embodiments, the length of the nucleic acid probe of the present invention may be 16 to 20 bases or 16 to 18 bases. By using probes of such length, Chlamydia pneumoniae can be detected with higher sensitivity.

[0043] In certain preferred embodiments, specific examples of the nucleic acid probes of the present invention include probes containing a base sequence S1 of at least 10 consecutive bases in the base sequence 35 to 70 of SEQ ID NO: 2 or SEQ ID NO: 3 (preferably SEQ ID NO: 2) or its complementary base sequence, or a base sequence S2 in which 1 to 3 bases are substituted, deleted, inserted, or added in base sequence S1. Probes can also include probes containing a base sequence S11 represented by any of SEQ ID NOs: 8 to 13 or its complementary base sequence, or a base sequence in which 1 to 3 bases (e.g., 1 or 2, or 1) are substituted, deleted, inserted, or added in base sequence S11. By using probes having such specific base sequences, it is possible to detect Chlamydia pneumoniae with even greater sensitivity.

[0044] Preferably, the above probe is labeled only at either the 5' end or the 3' end. In one embodiment, the nucleic acid probe of the present invention is preferably labeled such that it produces quenching or fluorescence when bound to a nucleic acid containing a base sequence that exhibits 85% or more, preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity with a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably labeled to produce quenching. There are no particular restrictions on the labeling substance, but a fluorescent dye is more preferable.

[0045] The fluorescent dye may be either a fluorescent substance that produces fluorescence by hybridizing with the target nucleic acid amplification product to form a complex, or a fluorescent substance that produces quenching. Preferably, it is a fluorescent substance that produces quenching when hybridized with the target nucleic acid amplification product, and more preferably, it is a fluorescent quenching dye that produces quenching through interaction with guanine during hybridization with the target nucleic acid amplification product (for example, a fluorescent quenching dye that produces quenching through interaction with guanine). Specifically, examples include, but are not limited to, at least one fluorescent quenching dye selected from the group consisting of fluoroceine and its derivatives (e.g., fluoroceine isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665).

[0046] More specifically, examples of fluorescent quenching dyes that quench by interaction with guanine include at least one selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G (CR6G), TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescent quenching dyes can be suitably used in the present invention.

[0047] In certain preferred embodiments, probes in which the terminal base labeled with a fluorescent quenching dye is cytosine are more preferred. Such probes can be quenched by forming base pairs with guanine bases in nucleic acid amplification products upon hybridization, allowing for very simple measurement of changes in the fluorescence intensity of the reaction solution.

[0048] Furthermore, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair when the probe hybridizes, fluorescence can be quenched if the distance between those bases is close enough. For example, details are described in Japanese Patent Publication No. 5354216, and the present invention can also refer to this technology. That is, when the probe hybridizes, fluorescence can be quenched if, for example, the guanine bases in the nucleic acid amplification product are within a range of 1 to 3 bases relative to the cytosine base of the probe (with the base that forms a base pair with the cytosine base being defined as 1).

[0049] Therefore, even if the nucleic acid probe has at least one terminal base other than cytosine that is labeled with a fluorescent quenching dye, the change in the fluorescence intensity of the reaction solution can be measured. For example, the details are described in Japanese Patent Publication No. 5354216, and the present invention can also refer to this technology. For example, when the probe hybridizes, if the guanine bases in the nucleic acid amplification product are present within a range of, for example, 1 to 3 bases relative to the terminal base labeled with the fluorescent quenching dye, quenching can occur (the base that forms a base pair with the terminal base is defined as 1).

[0050] In a particular preferred embodiment, the nucleic acid probe of the present invention is used in step (3). By using the nucleic acid probe of the present invention in this way, Chlamydia pneumoniae can be detected. The method of the present invention may use one type of probe or two or more types in combination.

[0051] In one embodiment, step (3) may include at least one of the following steps (3-a), (3-b), and (3-c): (3-a) Simultaneously with step (2), a step to monitor the progress of the reaction in step (2) (nucleic acid amplification reaction) in real time by hybridizing the nucleic acid probe of the present invention with the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution. (3-b) After the completion of step (2), the nucleic acid probe of the present invention is hybridized with the nucleic acid amplification product in the reaction solution, and the fluorescence intensity of the reaction solution is measured to monitor the progress of the reaction in step (2) (nucleic acid amplification reaction) at an endpoint. (3-c) After the completion of step (2), a step of hybridizing the nucleic acid probe of the present invention with the nucleic acid amplification product in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution. Steps (3-a), (3-b), or (3-c) allow for the simple, rapid, and highly sensitive detection of the formation of a complex between the nucleic acid amplification product and the nucleic acid probe. Steps (3-a), (3-b), and (3-c) may be performed in combination; for example, both steps (3-a) and (3-b), or both steps (3-a) and (3-c) may be performed. In one embodiment, from the viewpoint of more rapidly detecting the nucleic acid amplification product, steps (3-b) or (3-c) are preferred. Step (3-c), i.e., the melting curve analysis method, is particularly preferred.

[0052] (Step (3-a)) Step (3-a) is a method for monitoring the progress of the nucleic acid amplification reaction in real time (so-called real-time PCR), and quantitative analysis is possible by comparing it with a control substance of known concentration.

[0053] (Step (3-b)) Step (3-b) allows for rapid detection of the target nucleic acid contained in the sample by monitoring the progress of the nucleic acid amplification reaction at an endpoint. Furthermore, the approximate amount of target nucleic acid can be estimated by comparing the fluorescence intensity at the endpoint. For example, the progress of a nucleic acid amplification reaction can be monitored at an endpoint by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is complete, the fluorescence intensity of the reaction solution can be measured and compared to the fluorescence intensity of the reaction solution before the reaction to confirm whether or not the target nucleic acid has been amplified. Alternatively, the presence or absence of the target nucleic acid in the sample can also be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. A control reaction solution is a reaction solution to which a sample known to be negative or positive has been added instead of the sample to be measured. While the progress of nucleic acid amplification reactions generally needs to be monitored in real time, for the purpose of faster and simpler detection, it is preferable to measure at an endpoint.

[0054] (Step (3-c)) In step (3-c), measuring the temperature dependence of fluorescence intensity specifically means measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. By taking the first derivative of the obtained fluorescence intensity with respect to temperature, the melting temperature (Tm value) specific to the nucleic acid probe used can be determined. Alternatively, the fluorescence intensity may be converted to fluorescence quenching rate or other values ​​as needed. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. Generally, the Tm value is the temperature at which the proportion of oligonucleotides that form a double helix with their complementary strand is equal to the proportion that are single-stranded. Since the Tm value is a value specific to the base sequence, melting curve analysis can be used as a method to analyze base sequence polymorphisms of target nucleic acids. Base sequence polymorphisms here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0055] For example, melting curve analysis is also applied to SNP analysis. When there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases mismatch when the probe hybridizes, so the Tm value is generally low. Therefore, by comparing the magnitude of the Tm values, single nucleotide polymorphisms (SNP analysis) can also be performed.

[0056] In another embodiment, the method of the present invention may further use a probe and / or primer set for detecting other respiratory disease causative bacteria to distinguish and detect Chlamydia pneumoniae and other respiratory disease causative bacteria that may be present in a sample. The other respiratory disease causative bacteria are not particularly limited, but may include, for example, Mycoplasma pneumoniae, Klebsiella pneumoniae, Streptococcus pneumoniae, Bordetella pertussis, Bordetella parapertussis, Chlamydia psittacosis, and preferably Mycoplasma pneumoniae.

[0057] A method for distinguishing and detecting Chlamydia pneumoniae and other respiratory disease-causing bacteria that may be present in a sample may include, for example, the following steps (1), (2), and (3): (1) A step of providing a sample that may contain Chlamydia pneumoniae and other respiratory disease causative bacteria, (2) A step of performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1), a primer set for detecting Chlamydia pneumoniae, and a primer set for detecting other respiratory disease causative bacteria, and (3) The step of detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes for detecting Chlamydia pneumoniae and probes for detecting other respiratory disease causative bacteria.

[0058] Other probes for detecting respiratory disease-causing bacteria are not particularly limited, but probes for detecting Mycoplasma pneumoniae are preferred. For example, the probe for detecting Mycoplasma pneumoniae is the probe described in Japanese Patent Application Publication No. 2014-42459, and is more preferably a probe having the base sequence represented by Sequence ID No. 25. Other primer sets for detecting respiratory disease-causing bacteria are not particularly limited, but a primer set for detecting Mycoplasma pneumoniae is preferred. For example, the primer set for detecting Mycoplasma pneumoniae is the primer set described in Japanese Patent Application Publication No. 2014-42459, and is more preferably a combination of a primer having the nucleotide sequence represented by SEQ ID NO: 23 and a primer having the nucleotide sequence represented by SEQ ID NO: 24.

[0059] [Reagents for detecting Chlamydia pneumoniae] In another embodiment, the present invention provides a reagent for detecting Chlamydia pneumoniae. The reagent preferably contains, in addition to the nucleic acid probe of the present invention described above, at least components necessary for nucleic acid amplification and detection. These necessary components can each be known. For example, the reagent of the present invention preferably contains at least a set of nucleic acid primers for PCR, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. Multiple sets of the nucleic acid primer set for PCR and the nucleic acid probe for detection may be included to amplify multiple regions of Chlamydia pneumoniae. The concentration of each component can be adjusted as appropriate, but for example, the nucleic acid probe is preferably 0.01 to 1 μM, and more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, it is preferable that each nucleic acid probe included in the probe set is within the above concentration range. The nucleic acid primer is preferably 0.01 to 10 μM. The DNA polymerase is preferably 0.01 to 1 U / μL, and more preferably 0.02 to 0.5 U / μL. Deoxyribonucleoside triphosphates (dNTPs) are preferably present in a concentration of 0.02 to 1 mM, and more preferably in a concentration of 0.1 to 0.5 mM. Inorganic salts such as magnesium salts are preferably present in a concentration of 0.1 to 10 mM, and more preferably in a concentration of 1 to 5 mM.

[0060] Furthermore, for the purpose of suppressing nonspecific amplification or promoting the reaction, the reagents of the present invention may contain additives known in the art. Examples of additives for suppressing nonspecific amplification include known anti-DNA polymerase antibodies and phosphates. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding proteins (SSBs), T4 gene 32 protein, tRNA, sulfur or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoin, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween20, Nonidet® P40, etc. Furthermore, in this invention, these additives may be used individually or in combination of two or more types.

[0061] In certain embodiments, the reagent of the present invention preferably includes an internal control to facilitate the determination of false negatives. By using the internal control in combination, it is easy to confirm that the nucleic acid amplification reaction is proceeding normally, reducing the risk of false negatives and enabling more accurate Chlamydia pneumonia testing. When using the internal control in combination with the melting curve analysis method, it is preferable that the detection temperature of the internal control and the detection temperature of the Chlamydia pneumonia detection probe of the present invention are separated by a certain amount (for example, 5°C or more). For example, when using the internal control and / or the Chlamydia pneumonia detection probe of the present invention in the melting curve analysis method, the detection temperatures can be determined by methods known in the art, and those skilled in the art can appropriately select and use those whose detection temperatures differ by, for example, 5°C or more.

[0062] In certain embodiments, the reagent of the present invention can simultaneously detect Chlamydia pneumoniae and other pathogens. Simultaneous detection of other respiratory infection-causing bacteria in addition to the reagent of the present invention enables faster multi-parameter testing. When detecting other respiratory infection-causing bacteria in combination using the melting curve analysis method, it is preferable to use different fluorescent quenching dyes. For example, when using the melting curve analysis method with other respiratory infection-causing bacteria and / or the Chlamydia pneumoniae detection probe of the present invention, the fluorescent quenching dye can be determined by methods known in the art, and those skilled in the art can appropriately select and use them.

[0063] In another embodiment, the reagent of the present invention may include, as described above, probes and primer sets for detecting other respiratory disease causative agents, preferably probes and primer sets for detecting Mycoplasma pneumoniae.

[0064] [Kit for detecting Chlamydia pneumoniae] In another embodiment, the present invention provides a kit for detecting Chlamydia pneumoniae. The kit of the present invention is not particularly limited as long as it comprises the nucleic acid probe or reagent of the present invention as described above and is configured to detect (including differentiate) Chlamydia pneumoniae. For example, the kit of the present invention may optionally include a reagent capable of detecting (including quantitatively determining) the presence of the target to be detected, and / or instructions for use describing how to use the kit. For example, the kit of the present invention may be provided in which the nucleic acid probe, components necessary for the nucleic acid amplification reaction, and components necessary for detecting the amplification product are sealed in the same container or in separate containers, and packaged in a single package, for example, and include information on how to use the kit. The kit of the present invention may also include a positive control solution and / or a negative control solution. In another embodiment, the kit of the present invention may include, as described above, probes and primer sets for detecting other respiratory disease causative agents, preferably probes and primer sets for detecting Mycoplasma pneumoniae. [Examples]

[0065] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0066] Test Example 1: Confirmatory test for the detection of Chlamydia pneumoniae (1-1) Method To search for nucleic acid probes that can specifically detect Chlamydia pneumoniae, 25 copies of Chlamydia pneumoniae DNA were used per reaction, and a total of six nucleic acid probes designed based on the nucleotide sequences shown in SEQ ID NOs: 8-13 were used to check for the presence or absence of nonspecific amplification and the detection of Chlamydia pneumoniae. Purified water was used as the negative sample for checking for the presence or absence of nonspecific amplification. The nucleic acid probes used were oligonucleotides synthesized according to conventional methods, consisting of the nucleotide sequences shown in SEQ ID NOs: 8-13 (only one end, either the 5' or 3' end, was labeled with CR6G. See Figures 6 and 7 for the labeling position on each probe). In addition, a set of nucleic acid primers consisting of the nucleotide sequences shown in SEQ ID NOs: 14-17 and a set of nucleic acid primers consisting of one of the nucleotide sequences shown in SEQ ID NOs: 18-22 were used as nucleic acid primer sets.

[0067] (1-2) Reaction solution A reaction solution containing the following components was prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) of a known sequence was also added to the reagent to confirm that nucleic acid amplification was performed successfully. Primers represented by any of the following 0.5 μM SEQ ID NOs: 14-17 3.0 μM primers represented by any of SEQ ID NOs. 18-22 0.4 μM probes indicated by one of the sequence numbers 8-13

[0068] (1-3) Reactions Using GENECUBE®, the reaction solution was reacted using the following temperature cycles, and the fluorescence intensity was measured in each cycle. 94℃ 30 seconds, 97°C 1 second - 58°C 3 seconds - 63°C 5 seconds (60 cycles)

[0069] (1-4) Results Figure 1 shows the detection graph obtained when 25 copies of Chlamydia pneumoniae were detected by nucleic acid amplification and melting curve analysis using the probe shown as Sequence ID No. 8 as a representative example. Similarly, the measurement results using the probe used in this example are summarized in Table 1. The fluorescence change rate (representing the extinction rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0070] As shown in the results in Table 1, it was found that Chlamydia pneumoniae could be detected with all probes. Furthermore, the probe shown in Sequence ID No. 11 was designed to contain a mismatched base, different from the base sequence shown in Sequence ID No. 2, near the center of the base sequence constituting the probe. From these results, it was confirmed that the Chlamydia pneumoniae detection probe of the present invention may contain a mismatched base. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed regardless of which probe was used.

[0071] [Table 1]

[0072] Test Example 2: Confirmatory test for detection of Chlamydia pneumoniae in low-copy organisms (2-1) Method To search for nucleic acid probes that can specifically detect Chlamydia pneumoniae, 15 copies of Chlamydia pneumoniae DNA were used per reaction, and three nucleic acid probes designed based on the nucleotide sequences shown in SEQ ID NOs: 8-9 and 12 were used to check for the presence or absence of nonspecific amplification and the detection of Chlamydia pneumoniae. Purified water was used as the negative sample for checking for the presence or absence of nonspecific amplification. The nucleic acid probes used were oligonucleotides synthesized according to conventional methods, consisting of the nucleotide sequences shown in SEQ ID NOs: 8-9 and 12 (only one end, either the 5' or 3' end, was labeled with CR6G. See Figures 6 and 7 for the labeling position on each probe). In addition, a set of nucleic acid primers consisting of the nucleotide sequence shown in SEQ ID NOs: 14 or 16 and a set of nucleic acid primers consisting of the nucleotide sequence shown in SEQ ID NOs: 18-19 or 21 were used as nucleic acid primer sets.

[0073] (2-2) Reaction solution A reaction solution containing the following components was prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) of a known sequence was also added to the reagent to confirm that nucleic acid amplification was performed successfully. Primers represented by either SEQ ID NO: 14 or 16 (0.5 μM) 3.0 μM primer represented by SEQ ID NO: 18, 19, or 21 0.4 μM probes indicated by SEQ ID NOs. 8, 9, and 12.

[0074] (2-3) Reaction Using GENECUBE®, the reaction solution was reacted using the following temperature cycles, and the fluorescence intensity was measured in each cycle. 94℃ 30 seconds, 97°C 1 second - 58°C 3 seconds - 63°C 5 seconds (60 cycles)

[0075] (2-4) Results Figure 2 shows the detection graph obtained when 15 copies of Chlamydia pneumoniae were detected by nucleic acid amplification and melting curve analysis using the probe shown as sequence number 8 as a representative example. Similarly, the measurement results using the probe used in this example are summarized in Table 2. The fluorescence change rate (representing the extinction rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0076] As shown in the results in Table 2, it was found that the probes designed could detect Chlamydia pneumoniae with high sensitivity. Furthermore, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed regardless of the probe used.

[0077] [Table 2]

[0078] Example 3: Primer combination test for detecting Chlamydia pneumoniae (3-1) Method To verify the combination of nucleic acid primers (primer sets) used with the probe of the present invention confirmed in Examples 1 and 2, verification was performed using 10 copies of DNA derived from Chlamydia pneumoniae per reaction. In this test, nucleic acids consisting of the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 12 were synthesized according to a conventional method as the nucleic acid probe (labeled with CR6G at the 3' end; see Figures 6 and 7). The nucleic acid primers used were a set consisting of a nucleic acid primer shown in either SEQ ID NO: 14 or 16 and a nucleic acid primer consisting of the nucleotide sequences shown in any of SEQ ID NOs: 18 to 22.

[0079] (3-2) Reaction solution A reaction solution containing the following components was prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.). An internal control (IC) of a known sequence was also added to the reagent to confirm that nucleic acid amplification was performed successfully. Primer represented by either 0.5 μM SEQ ID NO: 14 or SEQ ID NO: 16 3.0 μM primers represented by any of SEQ ID NOs. 18-22 0.4 μM probes indicated by SEQ ID NOs. 8 and 12

[0080] (3-3) Reaction Using GENECUBE®, the reaction solution was reacted using the following temperature cycles, and the fluorescence intensity was measured in each cycle. 94℃ 30 seconds, 97°C 1 second - 58°C 3 seconds - 63°C 5 seconds (60 cycles)

[0081] (3-4) Results Figure 3 shows the detection graph obtained when Chlamydia pneumoniae was detected by nucleic acid amplification and melting curve analysis using the probe indicated by Sequence ID No. 8 and the primers indicated by the combination of Sequence ID Nos. 14 and 18. Similarly, the measurement results using the nucleic acid probe used in this example are summarized in Table 3. The fluorescence change rate (representing the quenching rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0082] As shown in these results, when using the probe of the present invention, it is possible to detect Chlamydia pneumoniae in a sample regardless of the primer combination, and regardless of which primer set is used. It was suggested that the influence of the selected nucleic acid primer combination is small when using either probe SEQ ID NO: 8 or SEQ ID NO: 12.

[0083] [Table 3]

[0084] Example 4: Detection of Chlamydia pneumoniae in combination with Mycoplasma pneumoniae detection system (4-1) Method To confirm the effects of combining the detection of other respiratory infection-causing bacteria with the probes of the present invention confirmed in Examples 1-3, a Mycoplasma pneumoniae detection system was added as a representative, and the effects of combining the two were verified using 20 copies each of Chlamydia pneumoniae-derived DNA and Mycoplasma pneumoniae-derived DNA per reaction. In this test example, nucleic acids consisting of the nucleotide sequences shown in SEQ ID NOs. 8 and SEQ ID NOs. 12 were synthesized according to a conventional method as nucleic acid probes (3' end labeled with CR6G; see Figures 6 and 7). The nucleic acid primers used were a set of nucleic acid primers consisting of either SEQ ID NOs. 14 or 16 and either SEQ ID NOs. 18 or 21. As a Mycoplasma pneumoniae detection system, arbitrarily designed nucleic acid primers shown in SEQ ID NOs. 23-24 and the probe shown in SEQ ID NOs. 25 were used (3' end labeled with BODIPY-FL).

[0085] (4-2) Reaction solution A reaction solution containing the following components was prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.). <Detection of Chlamydia pneumoniae> Primers represented by either SEQ ID NO: 14 or 16 (0.5 μM) 3.0 μM Primer represented by either SEQ ID NO: 18 or 21 0.4 μM probes indicated by either SEQ ID NO: 8 or 12 <Mycoplasma pneumoniae> Primer shown in 0.5 μM SEQ ID NO: 23 Primer shown in 3.0 μM SEQ ID NO: 24 0.4 μM probe shown in Sequence ID No. 25

[0086] (4-3) Reaction Using GENECUBE®, the reaction solution was reacted using the following temperature cycles, and the fluorescence intensity was measured in each cycle. 94℃ 30 seconds, 97°C 1 second - 58°C 3 seconds - 63°C 5 seconds (60 cycles)

[0087] (4-4) Results Figure 4 shows the detection graph obtained when Chlamydia pneumoniae and Mycoplasma pneumoniae were detected by nucleic acid amplification and melting curve analysis using the probe indicated by Sequence ID No. 8 and the Mycoplasma pneumoniae detection system. Similarly, the measurement results using the nucleic acid probe used in this example are summarized in Table 4. The fluorescence change rate (representing the extinction rate in this example) was calculated using the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0088] [Table 4]

[0089] Example 5: Detection of Chlamydia pneumoniae in nasopharyngeal specimens after simple pretreatment. (5-1) Method Samples were measured by adding Chlamydia pneumoniae DNA to the supernatant obtained by centrifugation of negative nasopharyngeal specimens at 13,000 rpm, at a rate of 20 copies / test.

[0090] (5-2) Reaction solution A reaction solution containing the following components was prepared using GeneCube® Test Basic (manufactured by Toyobo Co., Ltd.). Primer shown in 0.5 μM SEQ ID NO: 14 3.0 μM Primer shown in SEQ ID NO: 18 The probe indicated by 0.4 μM SEQ ID NO: 8

[0091] (5-3) Reaction Using GENECUBE®, the reaction solution was reacted using the following temperature cycles, and the fluorescence intensity was measured in each cycle. 94℃ 30 seconds, 97°C 1 second - 58°C 3 seconds - 63°C 5 seconds (60 cycles)

[0092] (5-4) Results Figure 5 shows the detection graph obtained when a negative nasopharyngeal sample was centrifuged at 13,000 rpm, and Chlamydia pneumoniae DNA was added to the supernatant at a rate of 10 copies / test. It can be seen that Chlamydia pneumoniae can be detected with sufficient sensitivity when using the primers and probes of the present invention. The supernatant of the nasopharyngeal sample used in this test example did not undergo a nucleic acid purification process and therefore contains various biological contaminants such as proteins that inhibit the nucleic acid amplification reaction. From the results of this test example, it was confirmed that, according to the present invention, even samples that have been simply processed in this manner can be measured with high sensitivity without any problems. [Industrial applicability]

[0093] By using the probe and / or primer set, reagent, or kit of the present invention, for example, Chlamydia pneumoniae, which may be present in a sample, can be detected simply, rapidly, and with high sensitivity. Therefore, the present invention can be used not only for research purposes but also to enable early detection and treatment of Chlamydia pneumoniae, which will help prevent the spread of sexually transmitted infections and can greatly contribute to clinical diagnosis and environmental testing.

Claims

1. A probe for detecting the tyrp gene in Chlamydia pneumoniae.

2. The probe according to claim 1, having the following base sequence (A) or (B): (A) Oligonucleotides comprising a sequence S1 of at least 10 consecutive bases in the base sequence 35 to 70 of Sequence ID No. 2 or Sequence ID No. 3 or its complementary base sequence, or a base sequence S2 in which 1 to 3 bases are substituted, deleted, inserted, or added to base sequence S1. (B) Only the 5' end or the 3' end of the oligonucleotide in (A) is labeled.

3. The probe according to claim 2, wherein the length of the base sequence of (A) or (B) is 10 to 35 bases.

4. The probe according to claim 2, wherein the base sequence of (A) or (B) includes the base sequence shown in any of SEQ ID NOs: 8 to 13 or a complementary base sequence thereof.

5. The probe according to claim 2, wherein the label is a fluorescent dye label.

6. The probe according to claim 2, wherein the label is a fluorescent quenching dye that quenches when bound to a nucleic acid containing a base sequence that exhibits 90% or more identity with a base sequence complementary to the base sequence of the probe.

7. The probe according to claim 2, wherein the label is a fluorescent quenching dye that is quenched by interaction with guanine.

8. The probe according to claim 2, wherein the label is a fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.

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

10. The probe according to claim 2, wherein the labeled terminal base is cytosine.

11. A method for detecting Chlamydia pneumoniae that may be present in a sample using a probe according to any one of claims 1 to 10.

12. The following steps (1), (2), and (3): (1) A step of providing a sample that may contain Chlamydia pneumoniae, (2) A step of carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1), and The method according to claim 11, further comprising the step of detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes.

13. The method according to claim 12, wherein step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.

14. The method according to claim 13, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a variant thereof.

15. The method according to claim 12, wherein the primer set for detecting Chlamydia pneumoniae used in the nucleic acid amplification reaction of step (2) comprises a first primer having a base sequence S3 of at least 15 consecutive bases in the base sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a complementary base sequence thereof, or a base sequence S5 or base sequence S4 in which 1 to 3 bases are substituted, deleted, inserted or added in base sequence S3, and a second primer having a base sequence S6 in which 1 to 3 bases are substituted, deleted, inserted or added in the base sequence S5 of SEQ ID NO: 6 or SEQ ID NO: 7 or a complementary base sequence thereof, and the second primer is complementary to the DNA extension product of the first primer.

16. The method according to claim 15, wherein the first primer has a base sequence shown in any of SEQ ID NOs: 14 to 17 or a complementary base sequence thereto, or a base sequence in which 1 to 3 bases are substituted, deleted, inserted, or added in that base sequence, and the second primer has a base sequence shown in any of SEQ ID NOs: 18 to 22 or a complementary base sequence thereto, or a base sequence in which 1 to 3 bases are substituted, deleted, inserted, or added in that base sequence.

17. A method for distinguishing and detecting Chlamydia pneumoniae and other respiratory disease-causing bacteria that may be present in a sample, using a probe set comprising the probe described in any one of claims 1 to 10 and a probe for detecting other respiratory disease-causing bacteria.

18. The following steps (1), (2), and (3): (1) A step of providing a sample that may contain Chlamydia pneumoniae and other respiratory disease causative bacteria, (2) A step of performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1), a primer set for detecting Chlamydia pneumoniae, and a primer set for detecting other respiratory disease causative bacteria, and The method according to claim 17, further comprising the step of (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probe sets.

19. The aforementioned probe for detecting other respiratory disease-causing bacteria has the base sequence represented by Sequence ID No. 25, The method according to claim 18, wherein the primer set for detecting other respiratory disease causative bacteria is a combination of a primer having the nucleotide sequence represented by SEQ ID NO: 23 and a primer having the nucleotide sequence represented by SEQ ID NO:

24.

20. The method according to claim 12, wherein the detection step in step (3) is performed by melting curve analysis.

21. A reagent or kit for detecting Chlamydia pneumoniae, comprising the probe described in any one of claims 1 to 10.

22. A reagent or kit for detecting Chlamydia pneumoniae, comprising the primer set according to claim 15 or 16.

23. A reagent or kit for distinguishing and detecting Chlamydia pneumoniae from other respiratory disease-causing bacteria, comprising the probe according to any one of claims 1 to 10, a primer set for detecting Chlamydia pneumoniae, a probe for detecting other respiratory disease-causing bacteria, and a primer set for detecting other respiratory disease-causing bacteria.

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