Methods for detecting pathogenic Chlamydia species

Abstract

To provide nucleic acid primer sets for differentially detecting Chlamydophila species and methods for detecting Chlamydophila species using the primer sets.SOLUTION: The invention provides a nucleic acid primer set for differentially detecting Chlamydophila species, the set comprising a nucleic acid primer comprising a sequence of consecutive 15-30 bases in a base sequence having 90% or more identity to a part of the genome sequence of ompA gene encoding a major outer membrane protein (MOMP) of Chlamydophila pneumonia or Chlamydophila psittaci, and a nucleic acid primer comprising a sequence of consecutive 15-30 bases in a base sequence complementary to the base sequence having 90% or more identity to the part of the genome sequence of ompA gene encoding the major outer membrane protein (MOMP) of Chlamydophila pneumonia or Chlamydophila psittaci. The invention also provides a method for detecting Chlamydophila species using the primer set.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide for distinguishing and detecting bacteria of the genus Chlamydophila contained in a specimen sample.Furthermore, the present invention relates to a method for detecting bacteria of the genus Chlamydophila contained in a specimen sample using the oligonucleotide, and a reagent / kit for use in the method. [Background technology]

[0002] Among Chlamydophila bacteria, Chlamydophila pneumoniae is the main causative agent of Chlamydial pneumonia, a highly contagious respiratory infection. Unlike Mycoplasma pneumonia, Chlamydophila pneumoniae is common not only in children but also in the elderly, and co-infection with other bacteria is not uncommon.

[0003] Chlamydial pneumonia is known to be caused by Chlamydophila pneumoniae, C. trachomatis, and C. psittaci.

[0004] C. trachomatis pneumonia is seen in newborns and infants, but in adults it is primarily a sexually transmitted disease that spreads through the pharynx and rarely leads to pneumonia except in those with weakened immune systems.

[0005] Chlamydia pneumoniae causes respiratory infections in humans and is responsible for approximately 10% of community-acquired pneumonia cases. Unlike mycoplasma pneumonia, it is not uncommon for it to occur in the elderly as well as in children, and it is often accompanied by co-infection with other bacteria.

[0006] Chlamydia psittacosis mainly causes respiratory infections. It is a zoonotic disease and is known as the causative agent of "psittacosis," which is transmitted from birds to humans. Although it is not common in community-acquired pneumonia, it is necessary to differentiate between moderate to severe atypical pneumonia and severe pneumonia of unknown cause.

[0007] In addition to Chlamydia pneumoniae, Chlamydia psittacosis, and Chlamydia trachomatis pneumonia, other microorganisms that cause pneumonia include Streptococcus pneumoniae, Mycoplasma pneumoniae, and Haemophilus influenzae. There is a strong demand for a test method that can specifically distinguish the causative bacteria of pneumonia from among these microorganisms and detect them simply, quickly, and with high sensitivity in clinical diagnosis.

[0008] Conventional methods for testing Chlamydophila bacteria include culture testing, serological testing, and genetic testing. Culture testing has problems in that it is difficult to isolate the bacteria and the detection sensitivity is low. Serological testing requires paired serum for accurate diagnosis, but at present this is not possible, and even if paired serum could be collected, it takes time to make a diagnosis. As for genetic testing, a method for detecting only Chlamydia pneumoniae using the PCR method has been proposed (for example, Patent Document 1).

[0009] In addition, methods for distinguishing and detecting Chlamydia pneumoniae from Chlamydia psittaci have been proposed (e.g., Patent Document 2). However, there is a need for the development of a more useful method for distinguishing and detecting Chlamydophila spp. with higher sensitivity and speed. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Publication No. 8-294400 [Patent Document 2] Japanese Patent Application Publication No. 9-121897 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention has been made against the background of the problems of the conventional technology. That is, the object of the present invention is to provide a method for easily and sensitively detecting Chlamydophila bacteria contained in a specimen sample. The present inventors have repeatedly studied conventionally known methods for distinguishing and detecting Chlamydia pneumoniae from Chlamydia psittaci, and have found that when a sample with insufficient nucleic acid purification is used, the method is susceptible to amplification inhibition due to the influence of substances contained in the specimen sample, and may not be able to detect accurately. Therefore, the present invention aims to provide a new method for easily and sensitively distinguishing and detecting Chlamydophila bacteria even when a sample with insufficient nucleic acid purification is used. [Means for solving the problem]

[0012] As a result of intensive research, the present inventors discovered that the use of specific oligonucleotides makes it possible to easily and sensitively detect Chlamydophila bacteria contained in a specimen sample, and thus arrived at the present invention. That is, the outline of the present invention is as follows.

[0013] [Item 1] A nucleic acid primer set for distinguishing and detecting Chlamydophila bacteria, comprising a nucleic acid primer corresponding to the following (I) and a nucleic acid primer corresponding to the following (II): (I) a nucleic acid primer consisting of a nucleotide sequence of 15 to 30 consecutive nucleotides that shows 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (II) A nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a base sequence complementary to a base sequence showing 90% or more identity to the base sequence shown in SEQ ID NO:1 or SEQ ID NO:2. [Item 2] The nucleic acid primer set according to Item 1, wherein the nucleic acid primers according to (I) and / or (II) above comprise a base sequence of 15 to 30 consecutive bases in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, or a base sequence in which one or several bases have been substituted, deleted, or added in said base sequence. [Item 3] The nucleic acid primer set according to Item 1 or 2, wherein the nucleic acid primer according to (I) is a nucleic acid primer consisting of a base sequence that shows 90% or more identity to a base sequence of 15 to 30 consecutive bases in the base sequence from base 1 to base 110 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the nucleic acid primer according to (II) is a nucleic acid primer consisting of a base sequence that shows 90% or more identity to a base sequence of 15 to 30 consecutive bases in a base sequence complementary to the base sequence from base 70 to base 220 of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. [Item 4] A nucleic acid primer set according to any one of Items 1 to 3, wherein the nucleic acid primer according to (I) is a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in the base sequence from the 1st to the 110th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence in which one or several bases have been substituted, deleted or added in said base sequence, and the nucleic acid primer according to (II) is a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a base sequence complementary to the 70th to 220th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence in which one or several bases have been substituted, deleted or added in said base sequence. [Item 5] A nucleic acid primer set according to any one of Items 1 to 4, which is used for distinguishing and detecting Chlamydophila pneumoniae and Chlamydophila psittaci as bacteria of the genus Chlamydophila. [Item 6] The nucleic acid primer set according to any one of Items 1 to 5, wherein the nucleic acid primer according to (I) above is a primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 3 to 7. [Item 7] The nucleic acid primer set according to any one of Items 1 to 6, wherein the nucleic acid primer according to (II) above is a primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 8 to 14. [Item 8] A method for distinguishing and detecting Chlamydophila bacteria that may be contained in a specimen, comprising the following steps (1) and (2): (1) performing a nucleic acid amplification reaction in a specimen using the nucleic acid primer set according to any one of Items 1 to 7 to generate a nucleic acid amplification product; and (2) A step of detecting the amplification product obtained in the step (1). [Item 9] A method for distinguishing and detecting bacteria of the genus Chlamydophila according to Item 8, wherein the step (2) comprises the following steps (2-1) to (2-2): (2-1) hybridizing the nucleic acid amplification product obtained in the step (1) with a nucleic acid probe to form a complex; and (2-2) A step of detecting the complex obtained in the step (2-1). [Item 10] The detection method described in Item 8 or 9, which distinguishes and detects C. pneumoniae and C. psittaci as Chlamydophila bacteria. [Item 11] The detection method according to any one of Items 9 and 10, wherein the nucleic acid probe used in the step (2-1) is a nucleic acid probe having the following characteristics (A) and (B): (A) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, which contains a base sequence of at least 15 consecutive bases; and (B) At least one of the terminal bases is labeled with a fluorescent quenching dye that quenches fluorescence upon interaction with guanine. [Item 12] The detection method according to Item 11, wherein the base sequence of at least 15 bases described in (A) is a base sequence of at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, or a base sequence in which one or several bases have been substituted, deleted, or added in such a base sequence. [Item 13] The detection method according to Item 11 or 12, wherein the base sequence of at least 15 bases described in (A) is a base sequence that shows 85% or more identity to a base sequence of at least 15 consecutive bases in the base sequence from the 60th to the 170th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto. [Item 14] The detection method according to any one of Items 11 to 13, wherein the base sequence of at least 15 bases described in (A) is a base sequence of at least 15 consecutive bases in the base sequence from the 60th to 170th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence in which one or several bases have been substituted, deleted, or added in such a base sequence. [Item 15] The detection method according to any one of Items 11 to 14, wherein the base sequence of at least 15 bases described in (A) consists of a base sequence shown in any one of SEQ ID NOs: 15 to 18. [Item 16] The detection method according to any one of Items 11 to 15, wherein the fluorescence quenching dye (B) is at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 17] The detection method according to any one of Items 11 to 16, wherein the fluorescence quenching dye (B) is at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 18] The detection method according to any one of Items 11 to 17, wherein in (B), at least one terminal base labeled with a fluorescence quenching dye is cytosine. [Item 19] The detection method according to any one of Items 8 to 18, wherein the step (2-2) includes at least one of the following steps (2-2a) to (2-2c): (2-2a) A step of hybridizing the nucleic acid probe to the obtainable amplification product and monitoring the progress of the nucleic acid amplification reaction in real time by measuring the fluorescence intensity of the reaction solution. (2-2b) After completion of the step (2-1), a step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the fluorescence intensity of the reaction solution to monitor the progress of the nucleic acid amplification reaction at an endpoint. (2-2c) A step of hybridizing the nucleic acid probe to the amplification product that can be obtained after completion of the step (2-1) and measuring the temperature dependence of the fluorescence intensity of the reaction solution. [Item 20] The detection method according to any one of Items 8 to 19, wherein the nucleic acid amplification reaction in the step (1) is PCR. [Item 21] The detection method according to any one of Items 8 to 20, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification reaction in the step (1) is a DNA polymerase belonging to Family B. [Item 22] The detection method according to Item 21, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Item 23] The detection method according to any one of Items 8 to 22, wherein the specimen sample is a biological sample that has not been subjected to a nucleic acid purification step. [Item 24] A nucleic acid probe used to distinguish and detect Chlamydophila bacteria that may be contained in a specimen sample, the nucleic acid probe having the following characteristics (A) and (B): (A) a base sequence showing 85% or more identity to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, which contains a base sequence of at least 15 consecutive bases; and (B) At least one of the terminal bases is labeled with a fluorescent quenching dye that quenches fluorescence upon interaction with guanine. [Item 25] The nucleic acid probe described in Item 24, wherein the base sequence of at least 15 bases described in (A) is a base sequence of at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, or a base sequence in which one or several bases have been substituted, deleted, or added in such a base sequence. [Item 26] The nucleic acid probe according to Item 24 or 25, wherein the base sequence of at least 15 bases described in (A) is a base sequence that shows 85% or more identity to a base sequence of at least 15 consecutive bases in the base sequence from the 60th to the 170th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto. [Item 27] ​​The nucleic acid probe according to any one of Items 24 to 26, wherein the base sequence of at least 15 bases described in (A) is a base sequence of at least 15 consecutive bases in the base sequence from the 60th to 170th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, or a base sequence in which one or several bases have been substituted, deleted or added in said base sequence. [Item 28] The nucleic acid probe according to any one of Items 24 to 27, which is used for distinguishing and detecting C. pneumoniae and C. psittaci as bacteria of the genus Chlamydophila. [Item 29] The nucleic acid probe according to any one of Items 24 to 28, wherein the base sequence of at least 15 bases described in (A) consists of a base sequence shown in any one of SEQ ID NOs: 15 to 18. [Item 30] The nucleic acid probe according to any one of Items 24 to 29, wherein the fluorescence quenching dye (B) is at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 31] The nucleic acid probe according to any one of Items 24 to 30, wherein the fluorescence quenching dye (B) is at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 32] The nucleic acid probe according to any one of Items 24 to 31, wherein in (B), at least one terminal base labeled with a fluorescence quenching dye is cytosine. [Item 33] A reagent for distinguishing and detecting bacteria of the genus Chlamydophila, comprising the nucleic acid primer set according to any one of Items 1 to 7 and / or the nucleic acid probe according to any one of Items 24 to 32. [Item 34] The reagent according to Item 33, which is used to distinguish and detect C. pneumoniae and C. psittaci as Chlamydophila bacteria. [Item 35] A kit for distinguishing and detecting bacteria of the genus Chlamydophila, comprising the nucleic acid primer set according to any one of Items 1 to 7, the nucleic acid probe according to any one of Items 24 to 32, and / or the reagent according to any one of Items 33 to 34. [Item 36] A kit according to Item 35 for distinguishing and detecting C. pneumoniae and C. psittaci as Chlamydophila bacteria. Effect of the Invention

[0014] According to the present invention, Chlamydophila bacteria contained in a specimen can be easily and sensitively detected and differentiated from the specimen, and Chlamydophila pneumoniae and Chlamydophila psittaci can be detected with high sensitivity. According to the method of the present invention, even when a sample in which nucleic acid has not been highly purified is used, it is difficult to be inhibited in amplification, and Chlamydophila bacteria can be detected and differentiated with sufficient sensitivity. By using the detection method, reagent, and kit using the oligonucleotide of the present invention, for example, Chlamydophila pneumoniae or Chlamydophila psittaci can be easily and sensitively detected, which can greatly contribute to the field of clinical diagnosis. [Brief description of the drawings]

[0015] [Figure 1]FIG. 1 shows the results of Example 1. [Diagram 2] FIG. 1 shows the results of Example 2. [Diagram 3] FIG. 1 shows the results of Example 3. [Figure 4] FIG. 1 shows the results of Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in more detail below while showing embodiments of the present invention, 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 addition, "~" in this specification means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In addition, "and / or" in this specification means either one or both.

[0017] In addition, in this specification, a nucleic acid primer may be referred to as an oligonucleotide primer or simply as a primer, and a nucleic acid probe may be referred to as an oligonucleotide probe or simply as a probe, and these are collectively referred to as oligonucleotides.

[0018] (1. Methods for distinguishing and detecting Chlamydophila spp.) One embodiment of the present invention is a method for distinguishing and detecting Chlamydophila bacteria that may be contained in a specimen. More specifically, the method of the present invention includes at least the following steps (1) and (2): (1) performing a nucleic acid amplification reaction in a specimen using a nucleic acid primer set including a nucleic acid primer having a specific base sequence to generate a nucleic acid amplification product; and (2) detecting the amplification product obtained in the step (1); That is, one feature of the present invention is that a nucleic acid primer set including a nucleic acid primer with a specific base sequence is used in a nucleic acid amplification reaction in order to obtain highly sensitive determination results in the detection of bacteria of the genus Chlamydophila.

[0019] [Nucleic acid primer set] The nucleic acid primer set according to (1) above, which is used in the present invention to distinguish and detect bacteria of the genus Chlamydophila, includes a nucleic acid primer corresponding to the following (I) and a nucleic acid primer corresponding to the following (II): (I) a nucleic acid primer consisting of a nucleotide sequence of 15 to 30 consecutive nucleotides that shows 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (II) A nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a base sequence complementary to a base sequence showing 90% or more identity to the base sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0020] SEQ ID NO: 1 as defined in (I) and (II) above is a nucleotide sequence corresponding to a part of the genome sequence of Chlamydophila pneumoniae, a type of Chlamydophila bacteria. SEQ ID NO: 2 is a part of the genome sequence of Chlamydophila parapertussis, a type of Chlamydophila bacteria. SEQ ID NO: 1 and SEQ ID NO: 2 are both known as the nucleotide sequences of the ompA gene encoding the major outer membrane protein (MOMP) in each of the Chlamydiae. The present invention is based on the discovery that Chlamydophila bacteria can be distinguished and detected with high sensitivity by performing a nucleic acid amplification reaction targeting the genomic region represented by the nucleotide sequence shown in SEQ ID NO: 1 (Chlamydophila pneumoniae) and / or the nucleotide sequence shown in SEQ ID NO: 2 (Chlamydophila psittaci).

[0021] It is known that the genomic sequences of Chlamydophila pneumoniae and Chlamydophila psittaci may differ slightly (for example, in one or several bases) depending on the strain. The present invention can also design the nucleic acid primer described in (I) or (II) above based on the genomic sequence of Chlamydophila pneumoniae or Chlamydophila psittaci that differs in one or several bases in this way. Therefore, the nucleic acid primer of the present invention specified in (I) or (II) above may be a nucleic acid primer consisting of a 15 to 30 consecutive base sequence in a base sequence that shows 90% or more identity with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto. From the viewpoint of expecting a more specific nucleic acid amplification reaction, the base sequence of the nucleic acid primer is preferably a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases that shows 93% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 100% identity to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto.

[0022] In a specific embodiment, the nucleic acid primer described in (I) or (II) above may be, for example, a nucleic acid primer consisting of a 15 to 30 consecutive base sequence in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto, or a base sequence in which one or several bases have been substituted, deleted, or added in the base sequence. Here, "one or several" may be, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2. In this way, by using a nucleic acid primer having a base sequence highly homologous to the base sequence shown in SEQ ID NO: 1 or 2, a more specific nucleic acid amplification reaction may be possible.

[0023] The nucleic acid primer used in the present invention may be any that can amplify the genome region of the Chlamydophila bacterium corresponding to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and may be a nucleic acid primer consisting of a nucleotide sequence of 15 to 30 consecutive bases in a region showing the same nucleotide sequence between SEQ ID NO: 1 and SEQ ID NO: 2, or may be a nucleic acid primer consisting of a nucleotide sequence of 15 to 30 consecutive bases in a region where some bases are different between SEQ ID NO: 1 and SEQ ID NO: 2. Furthermore, in the method of the present invention, a plurality of nucleic acid primers corresponding to the above (I) may be used, or a plurality of nucleic acid primers corresponding to the above (II) may be used. By using a plurality of primers corresponding to (I) and (II), a nucleic acid amplification reaction can also be performed by a multiplex method. From the viewpoint of enabling high-sensitivity detection at a lower cost, a method of performing a nucleic acid amplification reaction using a nucleic acid primer set containing 1 to 2 types of nucleic acid primers corresponding to the above (I) and 1 to 2 types of nucleic acid primers corresponding to the above (II) is preferred.

[0024] In one preferred embodiment, the nucleic acid primer defined in (I) above is a nucleic acid primer consisting of a 15-30 consecutive base sequence in a base sequence showing 90% or more identity with the 1st to 110th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary base sequence thereto. In this case, the nucleic acid primer defined in (II) above, which is preferably used in combination, is a nucleic acid primer consisting of a 15-30 consecutive base sequence in a base sequence showing 90% or more identity with the 70th to 220th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary base sequence thereto, and is designed to anneal to the 3'-end side of the site annealed by the nucleic acid primer defined in (I) above in the base sequence shown in SEQ ID NO: 1 or 2 or a complementary base sequence thereto. By generating a nucleic acid amplification product using such a combination of nucleic acid primers, it becomes possible to more effectively distinguish and detect Chlamydophila bacteria.

[0025] The length of the nucleic acid primers defined in (I) and (II) above is not particularly limited as long as it is 15 to 30 bases, but may be, for example, 20 to 30 bases. By using a nucleic acid primer of such a length, it is possible to suppress non-specific amplification reactions while enabling a sufficient nucleic acid amplification reaction, and to generate a specific nucleic acid amplification product with high sensitivity.

[0026] In a specific preferred embodiment, a specific example of the nucleic acid primer described in (I) above can be a nucleic acid primer having a base sequence shown in any one of SEQ ID NOs: 3 to 7. In a further specific preferred embodiment, a specific example of the nucleic acid primer described in (II) above can be a nucleic acid primer having a base sequence shown in any one of SEQ ID NOs: 8 to 13. In particular, by using a nucleic acid primer set including a combination of the nucleic acid primers described in (I) and (II), it is possible to distinguish and detect Chlamydophila bacteria with even higher sensitivity.

[0027] [Nucleic acid amplification reaction] In one embodiment, the method of the present invention uses the above-mentioned nucleic acid primer set to carry out a nucleic acid amplification reaction to generate a nucleic acid amplification product. The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level at which it can be visualized, i.e., to hundreds of millions of copies or more, and is widely used not only in the field of life science research, but also in the fields of clinical diagnosis, food hygiene inspection, environmental inspection, etc. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, TMA, etc. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method carried out in the present invention is preferably, but is not limited to, the PCR method (including the RT-PCR method).

[0028] [PCR reaction] PCR is a reaction catalyzed mainly by DNA polymerase, and the target nucleic acid is amplified by repeating three steps, namely (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase, as one cycle. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. In the present invention, it is preferable to use a DNA polymerase belonging to family B, from the viewpoint of enabling discrimination and detection of Chlamydophila bacteria more simply and sensitively.

[0029] The PCR reaction conditions are not particularly limited as long as the effects of the present invention are achieved, but for example, it is preferable to perform an initial heat deformation step at 80-100° C. for 0 seconds to 5 minutes, repeated heat deformation steps at 80-100° C. for 0.5-300 seconds, annealing at 35-80° C. for 1-300 seconds, and an extension reaction step at 35-85° C. for 1-300 seconds, and repeat this process 30-70 times. The temperature and time of the repeated cycles may be changed every one or several cycles.

[0030] [Family B DNA polymerase] The DNA polymerase used in the present invention is preferably, but is not limited to, a DNA polymerase belonging to family B. The DNA polymerase belonging to family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, more preferably a DNA polymerase derived from bacteria of the genus Pyrococcus and Thermococcus. The present invention also includes a mutant of an archaea-derived DNA polymerase belonging to family B that does not lose its activity. The DNA polymerase mutant includes, but is not limited to, a mutant for the purpose of enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, etc. Examples of DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants derived therefrom that have not lost their DNA polymerase activity. Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, and mutants thereof that have not lost their DNA polymerase activity. Preferably, DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) can be particularly preferably used in the present invention from the viewpoint of excellent extensibility and thermostability. PCR enzymes using these DNA polymerases are commercially available, and examples include 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.

[0031] [DNA polymerase derived from KOD] In this specification, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to Thermococcus kodakaraensis-derived DNA polymerase and its variants (for example, KOD-derived DNA polymerase in which 3'→5' exonuclease activity has been deleted by substituting, deleting, and / or adding one or several amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, the present invention performs a nucleic acid amplification reaction using such a KOD-derived DNA polymerase. KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and crude sample resistance compared to Taq DNA polymerase, which is a DNA polymerase belonging to family A. In the present invention, by using such a KOD DNA polymerase, it becomes possible to distinguish and detect Chlamydophila bacteria simply and with high sensitivity, as shown in the examples described later.

[0032] [Step of detecting the amplification product] In the method of the present invention, step (2) includes a step of detecting the amplification product obtained in step (1). The mode of this detection step is not particularly limited, and it can be carried out by any method known in the art.

[0033] In one embodiment, the step (2) includes the following steps (2-1) to (2-2): (2-1) hybridizing the nucleic acid amplification product obtained in the step (1) with a nucleic acid probe to form a complex; and (2-2) A step of detecting the complex obtained in the step (2-1). In a preferred embodiment, the present invention is characterized in that a nucleic acid probe capable of specifically reacting with the nucleic acid amplification product obtained in step (1) to form a complex is used in order to obtain highly sensitive determination results in the detection of bacteria of the genus Chlamydophila.

[0034] [Nucleic acid probe] In one embodiment, the present invention uses a nucleic acid probe capable of specifically reacting with a nucleic acid amplification product amplified with a nucleic acid primer set including a nucleic acid primer having the specific base sequence to form a complex.

[0035] The base sequence of the nucleic acid probe is preferably relatively highly homologous to the base sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 that can be amplified by the nucleic acid primers (I) and (II). For example, the nucleic acid probe used in the present invention may be a nucleic acid probe consisting of a base sequence showing 85% or more identity with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto, with at least 15 consecutive bases. Preferably, the nucleic acid probe may be a nucleic acid probe consisting of a base sequence of at least 15 consecutive bases in a base sequence showing 90% or more identity, more preferably 93% or more identity, even more preferably 95% or more identity, even more preferably 98% or more identity with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto. Particularly preferably, the nucleic acid probe is a nucleic acid probe consisting of a base sequence 100% identical to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence of at least 15 consecutive bases in a base sequence complementary thereto.

[0036] In a specific embodiment, the nucleic acid probe may be, for example, a nucleic acid probe consisting of a base sequence in which one or several bases have been substituted, deleted, or added in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Here, one or several may be, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2. By using a nucleic acid probe having a base sequence highly homologous to the base sequence shown in SEQ ID NO: 1 or 2, it becomes possible to distinguish and detect Chlamydophila bacteria with high sensitivity.

[0037] The nucleic acid probe of the present invention is not particularly limited as long as it has the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence highly homologous to a base sequence complementary thereto. However, when the nucleic acid probe is labeled with a fluorescent quenching dye that is quenched by interaction with guanine as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0038] In one preferred embodiment, the nucleic acid probe of the present invention is a nucleic acid probe consisting of a base sequence of at least 15 consecutive bases in a base sequence showing 85% or more identity with the 60th to 170th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence complementary thereto. By reacting such a nucleic acid probe with a nucleic acid amplification product, it becomes possible to more effectively distinguish and detect Chlamydophila bacteria.

[0039] The length of the nucleic acid probes defined in (I) and (II) above is not particularly limited as long as it is 15 bases or more, but may be, for example, 15 to 25 bases, preferably 16 to 19 bases. By using a nucleic acid probe of such a length, it is possible to more sensitively distinguish and detect Chlamydophila bacteria.

[0040] In a specific preferred embodiment, a specific example of the nucleic acid probe used in the present invention is a nucleic acid probe having a base sequence shown in any one of SEQ ID NOs: 15 to 18. By using a nucleic acid probe having such a specific base sequence, it becomes possible to distinguish and detect bacteria of the genus Chlamydophila with even higher sensitivity.

[0041] The nucleic acid probe may or may not be labeled. When labeled, the number of nucleic acids to be labeled is not particularly limited. Preferably, the nucleic acid at the end of the nucleic acid probe is labeled, and more preferably, only one of the nucleic acids at the end is labeled. There is no particular limit to the labeling substance, but it is more preferably a fluorescent substance.

[0042] The fluorescent label may be either a fluorescent substance that emits fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that is quenched by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that is quenched by interaction with guanine). Specific examples of the fluorescent quenching dye include, but are not limited to, at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665).

[0043] More specifically, examples of the fluorescence quenching dye that is quenched by interaction with guanine include at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

[0044] In a particularly preferred embodiment, a nucleic acid probe in which at least one terminal base labeled with a fluorescence quenching dye (that is, when both ends are labeled, at least one terminal base) is cytosine is more preferred. When such oligonucleotide probes hybridize to amplification products, they can quench the light by forming base pairs with the guanine bases in the amplification products and interacting with them, making it very easy to measure changes in the fluorescence intensity of the reaction solution.

[0045] In addition, when the probe is hybridized, even if the cytosine base of the probe and the guanine base in the amplified product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. That is, when the probe is hybridized, the fluorescence can be quenched if the guanine base in the amplified product is present within a range of, for example, 1 to 3 bases relative to the cytosine base of the probe (a base that forms a base pair with the cytosine base is considered to be 1).

[0046] In a further specific embodiment, even if at least one terminal base labeled with a fluorescence quenching dye (i.e., when both ends are labeled, at least one terminal base) is not cytosine in a nucleic acid probe, the change in fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. For example, when the probe hybridizes, quenching can be achieved if a guanine base in the amplification product is present within a range of, for example, 1 to 3 bases relative to at least one terminal base labeled with a fluorescence quenching dye (a base that forms a base pair with the terminal base is counted as 1).

[0047] [Step of detecting the complex] In the step of detecting the amplification product of the present invention, the embodiment of the step (2-2) of detecting a complex formed by hybridization of the nucleic acid amplification product with the nucleic acid probe is not particularly limited, and can be carried out by any method known in the art.

[0048] In one embodiment, the step (2-2) may include at least one of the following steps (2-2a) to (2-2c): (2-2a) A step of hybridizing the nucleic acid probe to the obtainable amplification product and monitoring the progress of the nucleic acid amplification reaction in real time by measuring the fluorescence intensity of the reaction solution. (2-2b) After completion of the step (2-1), a step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the fluorescence intensity of the reaction solution to monitor the progress of the nucleic acid amplification reaction at an endpoint. (2-2c) A step of hybridizing the nucleic acid probe to the amplification product that can be obtained after completion of the step (2-1) and measuring the temperature dependence of the fluorescence intensity of the reaction solution. According to the above steps (2-2a) to (2-2c), it may be possible to simply and sensitively detect the formation of a complex between a nucleic acid amplification product and a nucleic acid probe.

[0049] [(2-2a) Real-time monitoring of the progress of nucleic acid amplification reactions] By monitoring the progress of the nucleic acid amplification reaction in real time, the target nucleic acid contained in the sample can be rapidly detected, and further, the amount of the target nucleic acid contained in the sample can be quantified based on the amplification rate. For example, the progress of the nucleic acid amplification reaction is monitored in real time by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescent quenching dye. The fluorescence intensity of the reaction solution is measured at any time during the nucleic acid amplification reaction. If the sample contains DNA from Chlamydia pneumoniae or Chlamydia psittaci, the oligonucleotide probe hybridizes to the amplified nucleic acid, increasing (or decreasing) the fluorescence intensity depending on the amount of amplified nucleic acid. The progress of the nucleic acid amplification reaction can be monitored in real time by plotting the obtained fluorescence intensity against time (cycle number in the case of a PCR reaction).

[0050] [(2-2b) Monitoring the progress of nucleic acid amplification reactions at endpoints] By monitoring the progress of the nucleic acid amplification reaction at the end point, the target nucleic acid contained in the sample can be detected quickly, and the amount of the target nucleic acid can be quantified by comparing the fluorescence intensity at the end point. For example, the progress of the nucleic acid amplification reaction is monitored at an end point by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured. The presence or absence of amplification of the target nucleic acid can be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of the reaction solution before the reaction. Alternatively, the presence or absence of the target nucleic acid contained in the sample can also be confirmed by comparing the reaction intensity of the reaction solution after the reaction with the reaction intensity of a control reaction solution. A control reaction solution is a reaction solution to which a sample known to be negative or positive is added instead of the sample to be measured. It is generally preferable to monitor the progress of the nucleic acid amplification reaction in real time, but for the purpose of making the detection step more convenient, it is preferable to monitor it at an end point.

[0051] [(2-2c) Measuring the temperature dependence of fluorescence intensity] Measuring the temperature dependence of fluorescence intensity means, specifically, measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the oligonucleotide probe used can be obtained by first differentiating the obtained fluorescence intensity with respect to temperature. In addition, the fluorescence intensity may be converted into a fluorescence quenching rate or the like according to the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. In general, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that does not form a double strand and is single-stranded. Since the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms of target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0052] 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 oligonucleotide probe, the bases are mismatched when the probe hybridizes, and the Tm value is generally low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can be performed by comparing the magnitude of the Tm value.

[0053] [Specimen] The specimen sample that can be used in the present invention is not particularly limited as long as it may contain Chlamydophila bacteria. For example, oral scrapings, pharyngeal swabs, nasal swabs, nasal aspirates, sputum, bronchial lavage fluid, alveolar lavage fluid, etc. collected from subjects suspected of infection with C. pneumoniae or C. psittaci are included, but are not limited thereto. When a biological sample is used as the specimen sample to be measured, pretreatment such as dilution, suspension, centrifugation, or nucleic acid extraction may be performed depending on each biological sample, although this is not particularly limited.

[0054] The method of collecting and preparing the specimen is not particularly limited, and known methods can be used depending on the type and purpose of the sample. In a specific preferred embodiment, the specimen used in the present invention can be a sample obtained by treating a specimen collected from a living body with alkali (e.g., pH 8.0 to 14.0, preferably pH 10.0 to 13.0).

[0055] The method for extracting nucleic acid is not particularly limited, and any known method can be used depending on the type and purpose of the sample. For example, a kit sold by each manufacturer may be used for nucleic acid extraction.

[0056] In a specific preferred embodiment, the method of the present invention does not require a nucleic acid purification step that is usually considered essential in conventional nucleic acid amplification reactions. As shown in the results of the test examples described below, it has been confirmed that the method of the present invention can distinguish and detect Chlamydophila bacteria with high sensitivity even in specimen samples containing contaminants such as biological components other than the target nucleic acid (which may be inhibitors of the nucleic acid amplification reaction). In addition to the need for dedicated reagents, nucleic acid purification has the problem of complicated, time-consuming work. However, according to the gene amplification reaction of the present invention, there is no need to prepare a dedicated device for such nucleic acid purification, and it is possible to eliminate complicated operations. Furthermore, since the time required for centrifugation can be shortened, the time from collection of a biological sample from a subject to obtaining a genetic test result can be shortened, and for example, the time from collection of a sample to obtaining a genetic test result can be within one day, preferably within half a day, more preferably within 6 hours, even more preferably within 3 hours, and especially preferably within 2 hours (e.g., within one and a half hours). In this way, the method of the present invention, which can perform detection using biological samples that have not undergone a nucleic acid purification process, is preferable because it can eliminate the need for the labor of nucleic acid purification and enables the detection of Chlamydophila bacteria simply and quickly.

[0057] [Chlamydophila genus] The genus Chlamydophila is a gram-negative, obligately intracellular parasitic eubacterial coccobacillus known to be a causative agent of various respiratory diseases. Examples of Chlamydophila include C. pneumoniae and C. psittaci, which cause chlamydial pneumonia. According to the method of the present invention, any of these Chlamydophila can be detected with high sensitivity. Preferably, the method of the present invention can accurately detect and distinguish C. pneumoniae from C. psittaci.

[0058] [Reagents for distinguishing and detecting Chlamydophila species] Another embodiment of the present invention is a reagent for distinguishing and detecting Chlamydophila bacteria that may be contained in a sample. The reagent contains at least components necessary for nucleic acid amplification in addition to the nucleic acid primer set and / or nucleic acid probe of the present invention described above. The necessary components differ depending on the nucleic acid amplification reaction to be performed, and a known method can be used for each. For example, when detecting Chlamydophila bacteria that may be contained in a sample using a PCR reaction, it is preferable to contain at least DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. The concentration of each component can be appropriately adjusted depending on the intended experiment, and for example, the nucleic acid primer is preferably 0.01 to 10 μM. The nucleic acid probe is preferably 0.01 to 1 μM, more preferably 0.02 to 0.5 μM. The DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.02 to 0.5 U / uL. The concentration of deoxyribonucleoside triphosphates (dNTPs) is preferably 0.02 to 1 mM, and more preferably 0.1 to 0.5 mM. The concentration of inorganic salts such as magnesium salts is preferably 0.1 to 10 mM, and more preferably 1 to 5 mM.

[0059] Furthermore, additives known in the art may be added for the purpose of suppressing non-specific amplification or promoting the reaction. Known anti-DNA polymerase antibodies, phosphoric acid, etc. may be used as additives for suppressing non-specific amplification. Additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, Triton, Tween 20, and Nonidet P40. In the present invention, one or more of these additives may be used in combination, but is not limited to these.

[0060] [Kit for distinguishing and detecting Chlamydophila spp.] Another embodiment of the present invention is a kit for distinguishing and detecting Chlamydophila bacteria that may be contained in a sample. The kit of the present invention is not particularly limited as long as it contains the above-described nucleic acid primer set of the present invention, the nucleic acid probe, and / or a reagent containing them, and is configured to distinguish and detect Chlamydophila bacteria. For example, the kit of the present invention can optionally contain a genetic testing reagent that can detect or quantify the presence of the target to be detected and / or an instruction manual explaining the method of use, etc. For example, the nucleic acid primer set and the nucleic acid probe can be enclosed in the same container or in separate containers, and can be packaged in, for example, a single package and provided in a form including information on the method of use of the kit. EXAMPLES

[0061] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0062] Example 1: Detection of Chlamydophila sp. (1) Sample preparation DNA samples extracted from C. pneumoniae or C. psittaci were diluted with sterile water to 100 copies / μL each, and used as samples. (2) Nucleic acid amplification and melting curve analysis The above samples were added to the following reagents, and Chlamydia pneumoniae or Chlamydia psittaci was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.

[0063] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer represented by SEQ ID NO:3 0.25 μL 10 μM primer shown in SEQ ID NO: 8 1.5 μL 10 μM Probe represented by SEQ ID NO: 18 (labeled at the 3' end with BODIPY) 0.25 μL 3μL of sample

[0064] Nucleic acid amplification and melting curve analysis 94℃・2 minutes (One cycle) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 39℃~75℃ (Temperature rises at 0.09℃ / sec)

[0065] (3) Results Figure 1 shows the results of melting curve analysis of the change in fluorescence intensity with increasing temperature after nucleic acid amplification under the above conditions, with the horizontal axis representing temperature and the vertical axis representing the differential value of the fluorescence signal. In the graph, CPN genome represents the analysis results of Chlamydia pneumoniae genome DNA, CPS genome represents the analysis results of Chlamydia psittacosis genome DNA samples, and NC represents the analysis results of sterilized water. As is clear from Figure 1, it can be seen that Chlamydia pneumoniae and Chlamydia psittacosis are detected separately by the present invention.

[0066] Example 2: Detection of Chlamydia pneumoniae (1) Sample preparation A DNA sample (CPN genome) extracted from C. pneumoniae was mixed with a saline suspension of throat swab or postnasal swab to prepare a pseudo-biological sample at 10 copies / μL. This pseudo-biological sample was used as is without further nucleic acid extraction or purification, and therefore corresponds to a sample containing biological contaminants. (2) Nucleic acid amplification and melting curve analysis The above samples were added to the following reagents, and Chlamydia pneumoniae was detected under the following conditions: GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.

[0067] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer represented by SEQ ID NO:5 0.25 μL 10 μM primer shown in SEQ ID NO: 11 1.5 μL 10 μM Probe represented by SEQ ID NO: 15 (labeled with BODIPY at the 3' end) 0.25 μL 3μL of sample

[0068] Nucleic acid amplification and melting curve analysis 94℃・2 minutes (One cycle) 97℃・1 second 60℃・3 seconds 63℃・6 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 39℃~75℃ (Temperature rises at 0.4℃ / sec)

[0069] (3) Results Figure 2 shows the results of nucleic acid amplification and detection performed under the above conditions. The results are shown for the postnasal swab or pharyngeal swab to which Chlamydia pneumoniae genomic DNA was added. As shown in Figure 2, Chlamydia pneumoniae was detected in the postnasal swab and pharyngeal swab. This result revealed that, completely unexpectedly, Chlamydia pneumoniae can be detected without being inhibited by amplification contained in the specimen.

[0070] Example 3: Detection of Chlamydophila sp. (1) Sample preparation DNA samples extracted from C. pneumoniae or C. psittaci were diluted with sterile water to 15 copies / μL each, and used as samples. (2) Nucleic acid amplification and melting curve analysis The above samples were added to the following reagents, and Chlamydia pneumoniae or Chlamydia psittaci was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.

[0071] reagent A solution containing the following reagents was prepared: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 0.25μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer represented by SEQ ID NO:5 0.25 μL 10 μM primer represented by SEQ ID NO: 7 0.25 μL 10 μM Primer shown in SEQ ID NO: 11 1.0 μL 10 μM Primer shown in SEQ ID NO: 13 1.0 μL 10 μM Probe represented by SEQ ID NO: 15 (labeled with BODIPY at the 3' end) 0.25 μL 3μL of sample

[0072] Nucleic acid amplification and melting curve analysis 94℃・2 minutes (One cycle) 97℃・1 second 60℃・3 seconds 63℃・6 seconds (more than 60 cycles) 94℃・10 seconds 39℃・30 seconds 39℃~75℃ (Temperature rises at 0.15℃ / sec)

[0073] (3) Results 3 shows the results of melting curve analysis of the change in fluorescence intensity with increasing temperature after nucleic acid amplification under the above conditions, with the horizontal axis representing temperature and the vertical axis representing the differential value of the fluorescence signal. In the graph, CPN genome represents the analysis results of Chlamydia pneumoniae genome DNA, CPS genome represents the analysis results of Chlamydia psittaci genome DNA samples, and NC represents the analysis results of sterilized water. As is clear from FIG. 3, it can be seen that Chlamydia pneumoniae and Chlamydia psittaci are detected separately by the present invention.

[0074] Example 4: Detection of Chlamydia psittacosis (1) Sample preparation The throat swab was suspended in eSwab medium (manufactured by Copan), and a DNA sample (CPS genome) extracted from C. psittaci was mixed with the throat swab suspended in eSwab medium to prepare a concentration of 5 copies / μL, which was used as a pseudo-biological sample. This pseudo-biological sample was used as is without further nucleic acid extraction and purification, and therefore corresponds to a sample containing contaminating substances derived from living organisms. In addition, a DNA sample (CPS genome) extracted from C. psittaci was prepared with sterile water to a concentration of 5 copies / μL, which was used as a positive sample. (2) Nucleic acid amplification and melting curve analysis The above sample was added to the following example reagent and comparative reagent, and Chlamydia psittaci was detected under the following conditions: GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.

[0075] reagent A solution containing the following reagents was prepared as an example: KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PPD Mix (GeneCube(R) Test Basic, Toyobo) 1μL IC Mix (GeneCube(R) Test Basic, Toyobo) 1μL 10 μM primer represented by SEQ ID NO:6 0.25 μL 10 μM primer represented by SEQ ID NO: 14 1.5 μL 10 μM Probe shown in SEQ ID NO: 16 (5' end labeled with BODIPY, 3' end labeled with phosphorylation) 0.25 μL 3μL of sample

[0076] Furthermore, as a reagent for comparison with the above-mentioned reagent of this Example, a substantially identical reagent was prepared except that the following nucleic acid primer set (SEQ ID NOs: 19 to 20) was used, and used in a comparative experiment. 10 μM primer represented by SEQ ID NO: 19 0.25 μL 10 μM primer represented by SEQ ID NO: 20 1.5 μL

[0077] Nucleic acid amplification and melting curve analysis 94℃・2 minutes (One cycle) 97℃・1 second 58℃・3 seconds 63℃・6 seconds (more than 60 cycles) 94℃・30 seconds 39℃・30 seconds 39℃~75℃ (Temperature rises at 0.09℃ / sec)

[0078] (3) Results FIG. 4 shows the results of nucleic acid amplification and detection performed under the above conditions. In both the present embodiment and the comparative reagent, Chlamydophila psittaci was detected in the positive samples. On the other hand, in the pseudo-biological sample, the comparative reagent was unable to detect the amplification sufficiently due to the inhibition of amplification caused by substances contained in the specimen, whereas the present reagent was not inhibited and was able to detect with high sensitivity. From the results of this embodiment, it can be seen that by using the nucleic acid primer and nucleic acid probe of the present invention, the influence of impurities that may inhibit the amplification reaction can be suppressed and Chlamydophila genus can be detected with high sensitivity even in biological samples that have not been subjected to a nucleic acid purification process. [Industrial Applicability]

[0079] By using the nucleic acid primer set and / or nucleic acid probe of the present invention, it has become possible to easily and sensitively distinguish and detect Chlamydophila bacteria that may be contained in a sample. Therefore, even when a sample that is not sufficiently purified is used, the present invention makes it possible to easily and accurately distinguish and detect C. pneumoniae and C. psittaci, which have been difficult to rapidly distinguish in the past, and can greatly contribute to clinical diagnosis, etc.

Claims

1. A nucleic acid primer set for distinguishing and detecting Chlamydophila bacteria, which is a combination of a nucleic acid primer having the base sequence shown in SEQ ID NO:5 and a nucleic acid primer having the base sequence shown in SEQ ID NO:11, a combination of a nucleic acid primer having the base sequence shown in SEQ ID NO:7 and a nucleic acid primer having the base sequence shown in SEQ ID NO:13, or a combination of a nucleic acid primer having the base sequence shown in SEQ ID NO:6 and a nucleic acid primer having the base sequence shown in SEQ ID NO:

14.

2. The nucleic acid primer set according to claim 1, which is used for distinguishing and detecting Chlamydophila pneumoniae and Chlamydophila psittaci as bacteria of the genus Chlamydophila.

3. A method for distinguishing and detecting Chlamydophila bacteria that may be contained in a specimen, comprising the following steps (1) and (2): (1) performing a nucleic acid amplification reaction in a specimen sample using the nucleic acid primer set according to claim 1 or 2 to generate a nucleic acid amplification product; and (2) A step of detecting the amplification product obtained in the step (1).

4. The method for distinguishing and detecting Chlamydophila bacteria according to claim 3, wherein the step (2) comprises the following steps (2-1) to (2-2): (2-1) hybridizing the nucleic acid amplification product obtained in the step (1) with a nucleic acid probe having the base sequence shown in SEQ ID NO: 16 or 18 to form a complex; and (2-2) A step of detecting the complex obtained in the step (2-1).

5. The detection method according to claim 3 or 4, wherein, as Chlamydophila bacteria, C. pneumoniae and C. psittaci are detected in a distinguishable manner.

6. The method according to claim 4, wherein the nucleic acid probe used in the step (2-1) is a nucleic acid probe having the following characteristics (A) and (B): (A) consisting of the base sequence shown in SEQ ID NO: 16 or 18; and (B) At least one of the terminal bases is labeled with a fluorescent quenching dye that quenches fluorescence upon interaction with guanine.

7. The detection method according to claim 6, wherein the fluorescence quenching dye (B) is at least one fluorescence quenching dye selected from the group consisting of fluorescein, rhodamine, and BODIPY.

8. The detection method according to claim 6 or 7, wherein the fluorescence quenching dye (B) is at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5.7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

9. The detection method according to any one of claims 6 to 8, wherein in (B), at least one terminal base labeled with a fluorescent quenching dye is cytosine.

10. The detection method according to claim 4, wherein the step (2-2) includes at least one of the following steps (2-2a) to (2-2c): (2-2a) A step of monitoring the progress of the nucleic acid amplification reaction in real time by measuring the fluorescence intensity of a reaction solution containing or not containing a complex formed by hybridizing the nucleic acid probe to the nucleic acid amplification product. (2-2b) monitoring the progress of the nucleic acid amplification reaction at an endpoint by measuring the fluorescence intensity of a reaction solution that either contains or does not contain a complex formed by hybridizing the nucleic acid probe to the nucleic acid amplification product. (2-2c) A step of measuring the temperature dependence of the fluorescence intensity of a reaction solution containing or not containing a complex formed by hybridizing the nucleic acid amplification product with the nucleic acid probe.

11. The detection method according to any one of claims 3 to 10, wherein the nucleic acid amplification reaction in the step (1) is PCR.

12. 12. The method according to claim 3, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification reaction in the step (1) is a DNA polymerase belonging to family B.

13. The detection method according to claim 12, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a mutant thereof.

14. The detection method according to any one of claims 3 to 13, wherein the specimen sample is a biological sample that has not been subjected to a step of purifying nucleic acid.

15. A nucleic acid probe used for distinguishing and detecting Chlamydophila bacteria that may be contained in a specimen sample, the nucleic acid probe having the following characteristics (A) and (B): (A) consisting of the base sequence shown in SEQ ID NO: 16 or 18; and (B) At least one of the terminal bases is labeled with a fluorescent quenching dye that quenches fluorescence upon interaction with guanine.

16. The nucleic acid probe according to claim 15, which is used to distinguish and detect C. pneumoniae and C. psittaci as bacteria of the genus Chlamydophila.

17. 17. The nucleic acid probe according to claim 15, wherein the fluorescent quenching dye (B) is at least one fluorescent quenching dye selected from the group consisting of fluorescein, rhodamine, and BODIPY.

18. The nucleic acid probe according to any one of claims 15 to 17, wherein the fluorescent quenching dye (B) is 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).

19. The nucleic acid probe according to any one of claims 15 to 18, wherein in (B), at least one terminal base labeled with a fluorescent quenching dye is cytosine.

20. A reagent for distinguishing and detecting bacteria of the genus Chlamydophila, comprising the nucleic acid primer set according to claim 1 or 2 and / or the nucleic acid probe according to any one of claims 15 to 19.

21. The reagent according to claim 20, which is used to distinguish and detect C. pneumoniae and C. psittaci as bacteria of the genus Chlamydophila.

22. A kit for distinguishing and detecting Chlamydophila genus bacteria, comprising the nucleic acid primer set according to claim 1 or 2, the nucleic acid probe according to any one of claims 15 to 19, and / or the reagent according to claim 20 or 21.

23. The kit according to claim 22, for distinguishing and detecting C. pneumoniae and C. psittaci as bacteria of the genus Chlamydophila.

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