JP7608724B2 - Methods for detecting pathogenic Chlamydia species - Google Patents

Description

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

Chlamydophila bacteria, particularly Chlamydophila pneumoniae, are the main causative bacteria that cause 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.

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

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.

Chlamydia pneumoniae causes respiratory infections in many people and is responsible for approximately 10% of community-acquired pneumonia cases. Unlike mycoplasma pneumonia, it is not uncommon for the disease to develop in elderly people as well as children, and co-infection with other bacteria is not uncommon.

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

In addition to Chlamydia pneumoniae, Chlamydia psittacosis, and Chlamydia trachomatis pneumonia, other microorganisms that can cause pneumonia include Streptococcus pneumoniae, Mycoplasma pneumoniae, and Haemophilus influenzae. For clinical diagnosis, 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.

Conventional methods for testing Chlamydophila bacteria include culture testing, serological testing, and genetic testing. Culture testing has problems with the difficulty of isolating bacteria and low detection sensitivity. Serological testing requires paired serum for accurate diagnosis, but at present this is not possible, and even if paired serum could be obtained, 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).

Methods for distinguishing and detecting Chlamydia pneumoniae and Chlamydia psittacosis have also been proposed (e.g., Patent Document 2). However, there is a need for the development of a more useful method that can distinguish and detect Chlamydophila spp. more quickly and with higher sensitivity.

Japanese Patent Application Publication No. 8-294400 Japanese Patent Application Publication No. 9-121897

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 inventors have been studying conventionally known methods for distinguishing and detecting Chlamydia pneumoniae from Chlamydia psittacosis, and have found that this method is susceptible to amplification inhibition due to the influence of substances contained in the specimen sample when a sample in which nucleic acid has not been sufficiently purified is used, and accurate detection may not be possible. Therefore, the object of the present invention is to provide a new method for easily and sensitively distinguishing and detecting Chlamydophila bacteria even when a sample in which nucleic acid has not been sufficiently purified is used.

As a result of intensive research, the inventors discovered that Chlamydophila bacteria contained in a specimen can be detected easily and sensitively by using a specific oligonucleotide, and thus arrived at the present invention. That is, the outline of the present invention is as follows.

[Item 1] A nucleic acid primer set for distinguishing and detecting Chlamydophila genus 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 in a nucleotide sequence showing 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 primer according to (I) and/or (II) comprises 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] The nucleic acid primer set according to any one of Items 1 to 3, wherein the nucleic acid primer according to (I) above is a nucleic acid primer consisting of 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, 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) above is a nucleic acid primer consisting of 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, or a base sequence in which one or several bases have been substituted, deleted, or added in said base sequence.
[Item 5] The 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 base 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 base 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 sample, 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) detecting the amplification product obtained in the step (1).
[Item 9] A method for distinguishing and detecting Chlamydophila bacteria according to Item 8, wherein the step (2) comprises the following steps (2-1) to (2-2):
(2-1) a step of 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 according to Item 8 or 9, which distinguishes between 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) comprising a base sequence of at least 15 consecutive bases in 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; and (B) at least one terminal base is labeled with a fluorescent quenching dye that quenches by 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 the 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 the 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, tetrabromosulfonefluorescein (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 end point.
(2-2c) A step of hybridizing the nucleic acid probe to the amplification product 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 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) comprising a base sequence of at least 15 consecutive bases in 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; and (B) at least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.
[Item 25] The nucleic acid probe according to 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 said 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 base 60 to base 170 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 the base sequence.
[Item 28] The nucleic acid probe according to any one of Items 24 to 27, which is used to distinguish and detect 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, tetrabromosulfonefluorescein (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) above, 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 bacteria of the genus Chlamydophila.
[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] The kit according to Item 35, for distinguishing and detecting C. pneumoniae and C. psittaci as bacteria of the genus Chlamydophila.

According to the present invention, Chlamydophila bacteria contained in a specimen sample can be easily and sensitively detected, 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 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.

FIG. 1 shows the results of Example 1. FIG. 13 shows the results of Example 2. FIG. 13 shows the results of Example 3. FIG. 13 shows the results of Example 4.

The present invention will be described in further 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 the specification states "X~Y", 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.

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.

(1. Method for Differentiating and Detecting Chlamydophila Species)
One embodiment of the present invention is a method for distinguishing and detecting Chlamydophila bacteria that may be contained in a specimen sample. 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 of a specific base sequence is used in a nucleic acid amplification reaction in order to obtain a highly sensitive determination result in the detection of bacteria of the genus Chlamydophila.

[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 in a nucleotide sequence showing 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.

SEQ ID NO:1 defined in (I) and (II) above is a nucleotide sequence corresponding to a part of the genome sequence of Chlamydophila pneumoniae, a bacterium belonging to the genus Chlamydophila. SEQ ID NO:2 is a part of the genome sequence of Chlamydophila parapertussis, a bacterium belonging to the genus Chlamydophila. Both SEQ ID NO:1 and SEQ ID NO:2 are 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 genus bacteria can be distinguished and detected with high sensitivity by performing a nucleic acid amplification reaction targeting the genomic region represented by the base sequence shown in SEQ ID NO: 1 (Chlamydophila pneumoniae) and/or the base sequence shown in SEQ ID NO: 2 (Chlamydophila psittaci).

It is known that the genomic sequences of Chlamydophila pneumoniae and Chlamydophila psittaci may differ slightly (e.g., 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. Thus, the nucleic acid primer of the present invention as defined in (I) or (II) above may be a nucleic acid primer consisting of a contiguous base sequence of 15 to 30 bases 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.

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 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 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.

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 base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and may be a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a region showing the same base sequence between SEQ ID NO: 1 and SEQ ID NO: 2, or a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a region where some bases differ between SEQ ID NO: 1 and SEQ ID NO: 2. Furthermore, in the method of the present invention, multiple nucleic acid primers corresponding to the above (I) may be used, or multiple nucleic acid primers corresponding to the above (II) may be used. By using multiple primers corresponding to (I) and (II), a nucleic acid amplification reaction can also be performed by a multiplex method. From the viewpoint of enabling highly sensitive detection while being less expensive, 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.

In one preferred embodiment, the nucleic acid primer specified in (I) above is a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a base sequence showing 90% or more identity with the base sequence shown in the 1st to 110th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto. In this case, the nucleic acid primer specified in (II) above, which is preferably used in combination, is a nucleic acid primer consisting of a base sequence of 15 to 30 consecutive bases in a base sequence showing 90% or more identity with the base sequence shown in the 70th to 220th bases of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto, and is designed to anneal to the 3' end side of the site to which the nucleic acid primer of (I) above anneals in the base sequence shown in SEQ ID NO: 1 or 2 or a base sequence complementary 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 genus bacteria.

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 nucleic acid primers of such a length, it is possible to suppress non-specific amplification reactions while enabling a sufficient nucleic acid amplification reaction, and to generate specific nucleic acid amplification products with high sensitivity.

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.

[Nucleic acid amplification reaction]
In one embodiment, the method of the present invention uses a nucleic acid primer set as described above 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, and the like. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, and TMA. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method carried out in the present invention is preferably, but is not limited to, the PCR method (including the RT-PCR method).

[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 mutants. In view of being able to distinguish and detect Chlamydophila bacteria more simply and sensitively, it is preferable to use a DNA polymerase belonging to family B in the present invention.

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

[DNA polymerases belonging to family B]
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 the DNA polymerase derived from Archea belonging to family B that does not lose its activity. The mutant of DNA polymerase 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 polymerase derived from Thermococcus kodakaraensis and its mutants (for example, KOD-derived DNA polymerase lacking 3' to 5' exonuclease activity) can be particularly suitably used in the present invention from the viewpoint of excellent extensibility and thermal stability.
PCR enzymes using these DNA polymerases are commercially available, and 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 may be used in the present invention.

[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 a 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 is possible to distinguish and detect Chlamydophila genus bacteria in a simple yet highly sensitive manner, as shown in the examples described below.

[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 can be carried out by any method known in the art.

In one embodiment, the step (2) includes the following steps (2-1) to (2-2):
(2-1) a step of 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.

[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.

It is desirable that the base sequence of the nucleic acid probe has a relatively high homology with 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 or more consecutive bases. Preferably, it may be a nucleic acid probe consisting of a base sequence of at least 15 or more consecutive bases in a base sequence showing 90% or more, more preferably 93% or more, even more preferably 95% or more, and 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 preferred 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 or more consecutive bases in a base sequence complementary thereto.

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. In this way, 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.

The nucleic acid probe of the present invention is not particularly limited as long as it has a 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.

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 the nucleic acid amplification product, it becomes possible to more effectively distinguish and detect Chlamydophila bacteria.

The length of the nucleic acid probes specified 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.

In a specific preferred embodiment, a specific example of a 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 Chlamydophila bacteria with even higher sensitivity.

The nucleic acid probe may or may not be labeled. If it is labeled, there is no particular limit to the number of nucleic acids to be labeled. Preferably, the nucleic acid at the end of the nucleic acid probe is labeled, and more preferably, only one of the nucleic acids at either end is labeled. There is no particular limit to the labeling substance, but it is more preferably a fluorescent substance.

The fluorescent label may be either a fluorescent substance that fluoresces or a fluorescent substance that quenches by hybridizing with the target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches when hybridized with the target nucleic acid amplification product, and is particularly preferably a fluorescent quenching dye that quenches by interaction with guanine when hybridized with the target nucleic acid amplification product (e.g., a fluorescent quenching dye that quenches by interaction with guanine). Specifically, 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, BODI At least one fluorescent quenching dye selected from the group consisting of, but not limited to, PY-FL/C3, BODIPY-FL/C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, and BODIPY-665).

More specifically, examples of the fluorescent quenching dye that is quenched by interaction with guanine include 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, tetrabromosulfonefluorescein (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.

In a particularly preferred embodiment, a nucleic acid probe in which at least one terminal base labeled with a fluorescence quenching dye (i.e., 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.

When the probe hybridizes, 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 to each other. For example, details are described in Japanese Patent No. 5354216, and this technology can also be referenced in the present invention. In other words, when the probe hybridizes, the fluorescence can be quenched if the guanine base in the amplified product is within a range of, for example, 1 to 3 bases of the cytosine base of the probe (a base that forms a base pair with a cytosine base is counted as 1).

Furthermore, in certain embodiments, even if at least one terminal base labeled with a fluorescent quenching dye (i.e., if 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 this technology can also be referenced in the present invention. 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 of at least one terminal base labeled with a fluorescent quenching dye (the base that forms a base pair with the terminal base is counted as 1).

[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.

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 end point.
(2-2c) A step of hybridizing the nucleic acid probe to the amplification product 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.

(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. When the sample contains DNA of Chlamydia pneumoniae or Chlamydia psittacosis, 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).

(2-2b) End-point monitoring of the progress of nucleic acid amplification reactions
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.

[(2-2c) Measurement of temperature dependence of fluorescence intensity]
Specifically, the measurement of the temperature dependence of the fluorescence intensity means that the temperature of the reaction solution is changed from low to high while measuring the fluorescence intensity at each temperature. The melting temperature (Tm value) specific to the oligonucleotide probe used can be determined by first-order differentiation of the obtained fluorescence intensity with respect to temperature. The fluorescence intensity may also be converted into a fluorescence quenching rate or the like according to the purpose.
The detection, analysis, etc. of target nucleic acid using Tm value is called melting curve analysis. In general, 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 Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphism of target nucleic acid. Base sequence polymorphism here includes single base polymorphism, base substitution, base deletion, base insertion, etc.

For example, melting curve analysis is also applied to SNP analysis.
When the base sequence of the target nucleic acid is mutated with respect 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.

[Specimen sample]
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.

There are no particular limitations on the method of collecting and preparing the specimen sample, and known methods can be used depending on the type and purpose of the sample. In a particular preferred embodiment, the specimen sample 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).

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

In a particular preferred embodiment, the method of the present invention does not require a nucleic acid purification step, which 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 being complicated, time-consuming, and laborious. 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 six hours, even more preferably within three hours, and especially preferably within two hours (for example, within one and a half hours). In this way, the method of the present invention, which can perform detection using a biological sample that has not undergone a nucleic acid purification process, is preferable because it can eliminate the need for nucleic acid purification and allows for the detection of Chlamydophila bacteria simply and quickly.

[Chlamydophila spp.]
The genus Chlamydophila is a gram-negative, obligately intracellular, parasitic eubacterial coccobacillus known to be a causative agent of various respiratory diseases. Chlamydophila pneumoniae (C. pneumoniae) and Chlamydophila psittaci (C. psittaci) are known to cause pneumonia. According to the method of the present invention, any of these Chlamydophila genus bacteria can be detected with high sensitivity. Preferably, the method of the present invention is capable of accurately distinguishing and detecting C. pneumoniae from C. psittaci.

[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, but 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, more preferably 0.1 to 0.5 mM, and the concentration of inorganic salts such as magnesium salts is preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

Furthermore, additives known in the art may be added for the purpose of suppressing non-specific amplification or promoting the reaction. As additives for suppressing non-specific amplification, known anti-DNA polymerase antibodies, phosphoric acid, etc. may be used. As additives for promoting the reaction, 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, Nonidet P40, etc. may be mentioned. In the present invention, one or more of these additives may be used in combination, but is not limited to these.

[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 for use that explains the method of use, etc. For example, the nucleic acid primer set and the nucleic acid probe can be sealed in the same container or in separate containers, and can be packaged in, for example, a single package and provided in a form that includes information on the method of use of the kit.

The present invention will be specifically explained below based on examples. The present invention is not limited to the following examples.

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, 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 psittacosis was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.

Reagents A solution containing the following reagents was prepared:
KOD Mix (Genecube® Test Basic, Toyobo) 3 μL
PPD Mix (Genecube® Test Basic, Toyobo) 1 μL
IC Mix (Genecube® Test Basic, Toyobo) 1 μL
10 μM Primer shown in 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 with BODIPY at the 3' end) 0.25 μL
Sample 3 μL

Nucleic acid amplification and melting curve analysis 94℃・2 minutes (one cycle)
97℃・1 sec 58℃・3 sec 63℃・5 sec (60 cycles)
94°C for 30 seconds, 39°C for 30 seconds, 39°C to 75°C (temperature rises at 0.09°C/second)

(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 of the graph 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.

Example 2: Detection of Chlamydia pneumoniae
(1) Sample Preparation A DNA sample (CPN genome) extracted from C. pneumoniae was mixed with a pharyngeal swab or a postnasal swab suspended in saline 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 contaminants derived from a living organism.
(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.

Reagents A solution containing the following reagents was prepared:
KOD Mix (Genecube® Test Basic, Toyobo) 3 μL
PPD Mix (Genecube® Test Basic, Toyobo) 1 μL
IC Mix (Genecube® Test Basic, Toyobo) 1 μL
10 μM Primer shown in 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
Sample 3 μL

Nucleic acid amplification and melting curve analysis 94℃・2 minutes (one cycle)
97℃・1sec 60℃・3sec 63℃・6sec (60 cycles)
94°C for 30 seconds, 39°C for 30 seconds, 39°C to 75°C (temperature rises at 0.4°C/second)

(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 could be detected without being inhibited by amplification contained in the specimen.

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, 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 psittacosis was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.

Reagents A solution containing the following reagents was prepared:
KOD Mix (Genecube® Test Basic, Toyobo) 3 μL
PPD Mix (Genecube® Test Basic, Toyobo) 0.25 μL
IC Mix (Genecube® Test Basic, Toyobo) 1 μL
10 μM Primer shown in SEQ ID NO:5 0.25 μL
10 μM Primer shown in 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
Sample 3 μL

Nucleic acid amplification and melting curve analysis 94℃・2 minutes (one cycle)
97℃・1sec 60℃・3sec 63℃・6sec (60 cycles)
94°C for 10 seconds, 39°C for 30 seconds, 39°C to 75°C (temperature rises at 0.15°C/second)

(3) Results Figure 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 of the graph 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 3, it can be seen that Chlamydia pneumoniae and Chlamydia psittacosis are detected separately by the present invention.

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 pseudo-biological sample at 5 copies/μL. This pseudo-biological sample was used as is without further nucleic acid extraction and purification, and therefore corresponds to a sample containing contaminants derived from a living organism. In addition, a DNA sample (CPS genome) extracted from C. psittaci was prepared with sterile water at 5 copies/μL to prepare a positive sample.
(2) Nucleic Acid Amplification and Melting Curve Analysis The above sample was added to the following Example Reagent and Comparative Reagent, respectively, 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.

Reagents Solutions containing the following reagents were prepared as examples:
KOD Mix (Genecube® Test Basic, Toyobo) 3 μL
PPD Mix (Genecube® Test Basic, Toyobo) 1 μL
IC Mix (Genecube® Test Basic, Toyobo) 1 μL
10 μM Primer shown in SEQ ID NO:6 0.25 μL
10 μM Primer shown in SEQ ID NO: 14 1.5 μL
10 μM Probe represented by SEQ ID NO:16 (5' end labeled with BODIPY, 3' end labeled with phosphorylation) 0.25 μL
Sample 3 μL

In addition, as a reagent for comparison with the above-mentioned reagent of this embodiment, 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 comparison experiment.
10 μM Primer shown in SEQ ID NO: 19 0.25 μL
10 μM Primer shown in SEQ ID NO: 20 1.5 μL

Nucleic acid amplification and melting curve analysis 94℃・2 minutes (one cycle)
97℃・1sec 58℃・3sec 63℃・6sec (60 cycles)
94°C for 30 seconds, 39°C for 30 seconds, 39°C to 75°C (temperature rises at 0.09°C/second)

(3) Results Figure 4 shows the results of nucleic acid amplification and detection performed under the above conditions. In both the reagent of this embodiment and the comparative reagent, Chlamydia psittaci was detected in the positive samples. On the other hand, in the pseudo-biological sample, the comparative reagent was unable to detect Chlamydia psittaci sufficiently due to the inhibition of amplification caused by substances contained in the specimen, but it was revealed that the present reagent was not inhibited and could detect Chlamydophila 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 can be detected with high sensitivity even in biological samples that have not undergone a nucleic acid purification process.

By using the nucleic acid primer set and/or nucleic acid probe of the present invention, it is now 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 quickly distinguish in the past, and can make a significant contribution to clinical diagnosis, etc.

Claims (23)

A nucleic acid primer set for distinguishing and detecting Chlamydophila genus 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 . The nucleic acid primer set according to claim 1, which is used to distinguish and detect Chlamydophila pneumoniae and Chlamydophila psittaci as bacteria of the genus Chlamydophila. 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 claim 1 or 2 to generate a nucleic acid amplification product; and (2) detecting the amplification product obtained in the step (1). 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) detecting the complex obtained in the step (2-1). The detection method according to claim 3 or 4, which distinguishes between C. pneumoniae and C. psittaci as Chlamydophila bacteria. The method according to claim 4, wherein the nucleic acid probe used in the step (2-1) has 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 terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine. The detection method according to claim 6, wherein the fluorescent quenching dye (B) is at least one fluorescent quenching dye selected from the group consisting of fluorescein, rhodamine, and BODIPY. 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). 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. 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. The detection method according to any one of claims 3 to 10, wherein the nucleic acid amplification reaction in step (1) is PCR. The detection method according to any one of claims 3 to 11, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification reaction in step (1) is a DNA polymerase belonging to family B. 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. 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 process for purifying nucleic acid. 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 terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine. The nucleic acid probe according to claim 15, which is used to distinguish and detect C. pneumoniae and C. psittaci as Chlamydophila genus bacteria. The nucleic acid probe according to claim 15 or 16, wherein the fluorescent quenching dye (B) is at least one fluorescent quenching dye selected from the group consisting of fluorescein, rhodamine, and BODIPY. 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, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). 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. A reagent for distinguishing and detecting Chlamydophila bacteria, 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. The reagent according to claim 20, which is used to distinguish and detect C. pneumoniae and C. psittaci as Chlamydophila genus bacteria. A kit for distinguishing and detecting Chlamydophila 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. The kit according to claim 22 for distinguishing and detecting C. pneumoniae and C. psittaci as bacteria of the genus Chlamydophila.