JP7803376B2 - Method for detecting Bordetella spp. - Google Patents

Description

The present invention relates to oligonucleotides for distinguishing and detecting Bordetella bacteria contained in a test sample. Furthermore, the present invention relates to a method for detecting Bordetella bacteria contained in a test sample using the oligonucleotides, as well as reagents, kits, etc. for use in the method.

Bordetella bacteria, particularly Bordetella pertussis, are the primary causative agent of whooping cough, a highly contagious respiratory infection. Whooping cough can become particularly severe and even fatal when it infects infants who have not received the triple vaccine. Recently, whooping cough in previously vaccinated adults has become a problem due to a weakening of the vaccine's immune effectiveness. Because such infected adults rarely develop severe symptoms, they may not realize they are infected with Bordetella pertussis, which could spread the infection and become a source of infection for infants.

It is known that a portion of whooping cough cases (approximately 2-20%) are caused by Bordetella parapertussis. Although the symptoms of whooping cough caused by B. parapertussis are typically milder than those caused by B. pertussis, differential diagnosis based on clinical signs alone is difficult. Furthermore, it has been reported that existing methods for detecting B. pertussis may produce false-positive results when B. parapertussis is present in high concentrations (Non-Reference 1). Therefore, there is a strong need for a test method that can specifically distinguish between B. pertussis and B. parapertussis and that can detect them simply, quickly, and with high sensitivity for clinical diagnosis.

Conventional testing methods for Bordetella include culture testing, serological testing, and genetic testing. Culture testing has the problem of often being difficult to isolate the bacteria. Serological testing requires paired sera for accurate diagnosis, but these are currently unavailable, and even if paired sera are available, diagnosis takes time. Genetic testing, such as the Loop-Mediated Isothermal Amplification (LAMP) method, is known, but currently only detects Bordetella pertussis. Furthermore, the reaction system does not incorporate an internal control to confirm normal nucleic acid amplification, making it difficult to determine if amplification inhibition occurs. PCR-based methods for detecting Bordetella pertussis and Bordetella parapertussis are also being investigated, but they lack sufficient sensitivity and have the disadvantage of cross-reacting with other Bordetella pertussis and Bordetella species other than B. parapertussis (Non-Patent Document 1).

Furthermore, as an example of a method using genetic testing, methods have been proposed to detect Bordetella pertussis, which is one of the bacteria that cause respiratory infections, and distinguish it from other respiratory infections such as mycoplasma pneumonia and chlamydia pneumonia (e.g., Patent Document 1 and Patent Document 2).

Methods for distinguishing between Bordetella pertussis and Bordetella parapertussis have also been proposed (e.g., Patent Document 3). However, there is a need for the development of a more useful method that can distinguish between and detect Bordetella species with higher sensitivity.

Patent No. 6254085 Patent No. 4503844 JP 2014-230511 A

In Vitro Diagnostic Reagent FilmArray Respiratory Panel Package Insert (2nd Revised Edition, February 2019), bioMérieux Japan Co., Ltd.

The present invention was made in response to these problems in the prior art. Specifically, an object of the present invention is to provide a method for easily and sensitively detecting Bordetella bacteria contained in a specimen sample. The inventors have been studying previously known methods for distinguishing between Bordetella pertussis and Bordetella parapertussis, and have discovered that when using a sample in which nucleic acid has not been sufficiently purified, this method is susceptible to amplification inhibition due to the influence of substances contained in the sample, making accurate detection impossible. Therefore, an object of the present invention is to provide a new method for easily and sensitively distinguishing between Bordetella bacteria and Bordetella bacteria, even when using a sample in which nucleic acid has not been sufficiently purified.

As a result of extensive research, the inventors discovered that the use of specific oligonucleotides allows for simple and highly sensitive detection of Bordetella bacteria in test samples, leading to the present invention. The present invention is outlined below.

[Item 1] A nucleic acid primer set for distinguishing and detecting Bordetella 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 primers according to (I) and/or (II) above consist 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.
[Item 3] The nucleic acid primer set according to Item 1 or 2, which is used to distinguish and detect Bordetella pertussis and Bordetella parapertussis as bacteria of the genus Bordetella.
[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 primer consisting of a base sequence shown in any one of SEQ ID NOs: 3 to 9.
[Item 5] The nucleic acid primer set according to any one of Items 1 to 4, 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: 10 to 15.
[Item 6] A method for distinguishing and detecting Bordetella 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 sample using the nucleic acid primer set according to any one of Items 1 to 5 to generate a nucleic acid amplification product; and (2) detecting the amplification product obtained in the step (1).
[Item 7] A method for distinguishing and detecting Bordetella bacteria according to Item 6, 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) detecting the complex obtained in the step (2-1).
[Item 8] The detection method according to Item 6 or 7, wherein B. pertussis and B. parapertussis are detected separately as Bordetella bacteria.
[Item 9] The detection method according to any one of Items 6 to 8, 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 comprises a base sequence of at least 15 consecutive bases; and (B) at least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.
[Item 10] The detection method according to Item 9, 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 11] The detection method according to Item 9 or 10, wherein the base sequence of at least 15 bases described in (A) consists of the base sequence shown in any one of SEQ ID NOs: 16 to 20.
[Item 12] The detection method according to any one of Items 9 to 11, 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 13] The detection method according to any one of Items 9 to 12, 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 14] The detection method according to any one of Items 9 to 13, wherein in (B) at least one terminal base labeled with a fluorescence quenching dye is cytosine.
[Item 15] The detection method according to any one of Items 7 to 14, 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 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 in real time.
(2-2b) After completion of the step (2-1), the nucleic acid probe is hybridized to the amplification product that can be obtained, and the fluorescence intensity of the reaction solution is measured to monitor the progress of the nucleic acid amplification reaction at an endpoint.
(2-2c) After completion of the step (2-1), the nucleic acid probe is hybridized to the amplification product that can be obtained, and the temperature dependence of the fluorescence intensity of the reaction solution is measured.
[Item 16] The detection method according to any one of Items 6 to 15, wherein the nucleic acid amplification reaction in step (2) is PCR.
[Item 17] The detection method according to any one of Items 6 to 16, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification reaction in step (2) is a DNA polymerase belonging to family B.
[Item 18] The detection method according to Item 17, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof.
[Item 19] The detection method according to any one of Items 6 to 18, wherein the specimen sample is a biological sample that has not been subjected to a nucleic acid purification step.
[Item 20] A nucleic acid probe used to distinguish and detect Bordetella 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 comprises a base sequence of at least 15 consecutive bases; and (B) at least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.
[Item 21] The nucleic acid probe according to Item 20, wherein the base sequence of at least 15 bases described in (A) above 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 22] The nucleic acid probe according to Item 20 or 21, which is used to distinguish and detect B. pertussis and B. parapertussis as bacteria of the genus Bordetella.
[Item 23] The nucleic acid probe according to any one of Items 20 to 22, wherein the base sequence of at least 15 bases described in (A) consists of the base sequence shown in any one of SEQ ID NOs: 16 to 20.
[Item 24] The nucleic acid probe according to any one of Items 20 to 23, 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 25] The nucleic acid probe according to any one of Items 20 to 24, 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 26] The nucleic acid probe according to any one of Items 20 to 25, wherein in (B) above, at least one terminal base labeled with a fluorescence quenching dye is cytosine.
[Item 27] A reagent for differentially detecting bacteria of the genus Bordetella, comprising the nucleic acid primer set according to any one of Items 1 to 5 and/or the nucleic acid probe according to any one of Items 20 to 26.
[Item 28] The reagent according to Item 27, which is used to distinguish between B. pertussis and B. parapertussis as bacteria of the genus Bordetella.
[Item 29] A kit for differentially detecting bacteria of the genus Bordetella, comprising the nucleic acid primer set according to any one of Items 1 to 5, the nucleic acid probe according to any one of Items 20 to 26, and/or the reagent according to any one of Items 27 to 28.
[Item 30] The kit according to Item 29, for distinguishing between B. pertussis and B. parapertussis as bacteria of the genus Bordetella.

The present invention enables the simple and highly sensitive detection of Bordetella genus bacteria contained in a specimen sample, and in particular, enables the highly sensitive detection of Bordetella pertussis and Bordetella parapertussis. According to the method of the present invention, even when using a sample in which nucleic acid has not been highly purified, amplification inhibition is resistant and Bordetella genus bacteria can be detected with sufficient sensitivity. The use of detection methods, reagents, and kits using the oligonucleotides of the present invention enables the simple and highly sensitive detection of, for example, Bordetella pertussis or Bordetella parapertussis, making a significant contribution to the field of clinical diagnosis.

FIG. 1 shows the results of Example 1. FIG. 10 shows the results of Example 2. FIG. 10 shows the results of Example 3. FIG. 10 shows the results of Example 4. FIG. 1 shows the results of Example 6.

The present invention will be described in further detail below, showing embodiments thereof, but the present invention is not limited to these. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, the term "~" in this specification means "at least, at most." For example, "X to Y" in the specification means "at least X, at most Y." In addition, "and/or" in this specification means either one or both.

Furthermore, in this specification, nucleic acid primers may be referred to as oligonucleotide primers or simply as primers, and nucleic acid probes may be referred to as oligonucleotide probes or simply as probes, and these are collectively referred to as oligonucleotides.

(1. Method for distinguishing and detecting Bordetella species)
One embodiment of the present invention is a method for distinguishing and detecting Bordetella bacteria that may be contained in a test 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 sample 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 Bordetella.

[Nucleic acid primer set]
The nucleic acid primer set described in (1) above, which is used to distinguish and detect bacteria of the genus Bordetella in the present invention, 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, as defined in (I) and (II) above, is a nucleotide sequence corresponding to a portion of the genome sequence of Bordetella pertussis, a type of Bordetella bacterium. SEQ ID NO: 2 is a portion of the genome sequence of Bordetella parapertussis, a type of Bordetella bacterium, and is the nucleotide sequence of a region showing homology to a portion of the B. pertussis genome sequence shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 1 (Bordetella pertussis) and the nucleotide sequence shown in SEQ ID NO: 2 (Bordetella parapertussis) are genomic regions showing high homology (approximately 99.6%). The present invention is based on the discovery that Bordetella bacterium can be distinguished and detected with high sensitivity by performing a nucleic acid amplification reaction targeting this genomic region.

It is known that the genomic sequences of Bordetella pertussis and Bordetella parapertussis may differ slightly (e.g., in one or several bases) depending on the strain. The present invention also allows the design of the nucleic acid primer described in (I) or (II) above based on the genomic sequence of Bordetella pertussis or Bordetella parapertussis that differs by one or several bases. Therefore, the nucleic acid primer of the present invention described in (I) or (II) above may be a nucleic acid primer consisting of a 15 to 30 consecutive base sequence in a nucleotide sequence that shows 90% or greater identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto. From the perspective 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 certain embodiments, the nucleic acid primer described in (I) or (II) above may be, for example, a nucleic acid primer consisting of a 15-30 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 such a base sequence. Here, "one or several" may mean, for example, 1-5, preferably 1-4, more preferably 1-3, and even more preferably 1-2. In this way, using a nucleic acid primer having a base sequence highly homologous to the base sequence shown in SEQ ID NO: 1 or 2 may enable a nucleic acid amplification reaction with higher specificity.

The nucleic acid primers used in the present invention may be any that can amplify the genomic region of Bordetella bacteria corresponding to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. They may be nucleic acid primers consisting of a nucleotide sequence of 15 to 30 consecutive bases in a region showing identical nucleotide sequences between SEQ ID NO: 1 and SEQ ID NO: 2, or nucleic acid primers consisting of a nucleotide 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 (I) above may be used, or multiple nucleic acid primers corresponding to (II) above may be used. Nucleic acid amplification reactions can also be performed using a multiplex method by using multiple primers corresponding to (I) and (II). From the perspective of enabling highly sensitive detection at lower costs, a method of performing nucleic acid amplification reactions using a nucleic acid primer set containing one nucleic acid primer corresponding to (I) above and one nucleic acid primer corresponding to (II) above is preferred.

In one preferred embodiment, the nucleic acid primer specified in (I) above is a nucleic acid primer consisting of a 15-30 consecutive base sequence in the 70th to 190th bases of the nucleotide 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 that is preferably used in combination is a nucleic acid primer consisting of a 15-30 consecutive base sequence in the 220th to 340th bases of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 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 Bordetella bacteria.

In another preferred embodiment, the nucleic acid primer specified in (I) above is a nucleic acid primer consisting of a 15-30 consecutive base sequence in the 300th to 390th bases of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary nucleotide sequence thereto. In this case, the nucleic acid primer specified in (II) above that is preferably used in combination is a nucleic acid primer consisting of a 15-30 consecutive base sequence in the 470th to 510th bases of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary nucleotide sequence thereto. By generating nucleic acid amplification products using such a combination of nucleic acid primers, it is also possible to effectively distinguish and detect Bordetella bacteria.

The length of the nucleic acid primers specified in (I) and (II) above is not particularly limited as long as it is 15 to 30 bases, but it can be, for example, 16 to 25 bases, preferably 17 to 24 bases. Using nucleic acid primers of such lengths enables sufficient nucleic acid amplification reaction while suppressing nonspecific amplification reaction, making it possible to produce specific nucleic acid amplification products with high sensitivity.

In a particularly preferred embodiment, a specific example of the nucleic acid primer described in (I) above is a nucleic acid primer consisting of the base sequence shown in any of SEQ ID NOs: 3 to 9. In a further particularly preferred embodiment, a specific example of the nucleic acid primer described in (II) above is a nucleic acid primer consisting of the base sequence shown in any of SEQ ID NOs: 10 to 15. 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 Bordetella genus bacteria with even higher sensitivity.

[Nucleic acid amplification reaction]
In one embodiment, the method of the present invention involves performing a nucleic acid amplification reaction using the nucleic acid primer set described above to generate a nucleic acid amplification product. Nucleic acid amplification is a technique for amplifying a few copies of a target nucleic acid to a level at which it can be visualized, i.e., hundreds of millions of copies or more, and is widely used not only in the field of life science research but also in fields such as clinical diagnosis, food hygiene testing, and environmental testing. 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 used in the present invention is preferably PCR (including RT-PCR), but is not limited to this.

[PCR reaction]
The PCR reaction is primarily catalyzed by DNA polymerase, and involves three steps: (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using a DNA polymerase. This cycle is repeated to amplify the target nucleic acid. Examples of DNA polymerases 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, which allows for more convenient and sensitive detection of Bordetella species.

The PCR reaction conditions are not particularly limited as long as they achieve the effects of the present invention. For example, it is preferable to perform the initial heat transformation step at 80-100°C for 0 seconds to 5 minutes, the repeated heat transformation 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 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 or Thermococcus. The present invention also includes mutants of DNA polymerases derived from Archaea belonging to Family B that do not lose their activity. Examples of DNA polymerase mutants include, but are not limited to, mutants intended for enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, etc.
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 thereof derived therefrom that have not lost their DNA polymerase activity.
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, as well as 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'->5' exonuclease activity) are particularly suitable for use in the present invention because of their excellent extensibility and thermal stability.
PCR enzymes using these DNA polymerases are commercially available, including Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[KOD-derived DNA polymerase]
As used herein, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to DNA polymerase derived from Thermococcus kodakaraensis and its variants (e.g., KOD-derived DNA polymerase in which 3' to 5' exonuclease activity has been deleted by substituting, deleting, and/or adding one or more amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, the present invention uses such KOD-derived DNA polymerase to perform nucleic acid amplification reactions. KOD DNA polymerase has superior accuracy, amplification efficiency, extensibility, and crude sample resistance compared to Taq DNA polymerase, a DNA polymerase belonging to Family A. In the present invention, the use of such KOD DNA polymerase enables simple yet highly sensitive differentiation and detection of Bordetella spp., as shown in the examples described below.

[Step of detecting amplification products]
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.

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) 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 Bordetella.

[Nucleic acid probe]
In one embodiment, the present invention uses a nucleic acid probe that can specifically react with and form a complex with a nucleic acid amplification product amplified with a nucleic acid primer set containing a nucleic acid primer consisting of the specific base sequence.

The base sequence of the nucleic acid probe desirably has a relatively high homology to the base sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2, which can be amplified by the nucleic acid primers (I) and (II). For example, the nucleic acid probe used in the present invention may consist of 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, consisting of a base sequence of at least 15 consecutive bases. Preferably, the nucleic acid probe consists of a base sequence of at least 15 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 to 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 consecutive bases in a base sequence complementary thereto.

In certain embodiments, 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 mean, 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 Bordetella bacteria with high sensitivity.

The nucleic acid probe of the present invention is not particularly limited as long as it has a sequence highly homologous to the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto. However, when the nucleic acid probe is labeled with a fluorescent quenching dye that quenches fluorescence through 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 the base sequence shown in positions 190 to 220 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 Bordetella bacteria.

In another 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 the base sequence shown at positions 400 to 440 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 Bordetella 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 it can be, for example, 15 to 25 bases, preferably 16 to 19 bases. Using nucleic acid probes of such lengths makes it possible to more sensitively distinguish and detect Bordetella bacteria.

In a specific preferred embodiment, a specific example of a nucleic acid probe used in the present invention is a nucleic acid probe consisting of the base sequence shown in any of SEQ ID NOs: 16 to 20. By using a nucleic acid probe having such a specific base sequence, it becomes possible to distinguish and detect Bordetella bacteria with even higher sensitivity.

The nucleic acid probe described above may or may not have labeled nucleic acids. If labeled, there is no particular limit to the number of labeled nucleic acids. Preferably, the nucleic acid at the end of the nucleic acid probe is labeled, and more preferably, only one of the terminal nucleic acids is labeled. There are also no particular limits on the labeling substance, but a fluorescent substance is more preferred.

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. Particularly preferred are fluorescent quenching dyes that quench fluorescence by interacting with guanine when hybridized with a target nucleic acid amplification product (e.g., fluorescent quenching dyes that quench fluorescence by interacting with guanine). Specific examples include 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 Examples of such quenching dyes include, but are not limited to, at least one fluorescence quenching dye selected from the group consisting of 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, and BODIPY-665.

More specifically, examples of fluorescence quenching dyes that quench through 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.

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 form base pairs with the guanine bases in the amplification products and interact to quench the light, making it very easy to measure changes in the fluorescence intensity of the reaction solution.

When the probe hybridizes, fluorescence can be quenched even if the cytosine base in the probe and the guanine base in the amplified product do not form a base pair, as long as the bases are close to each other. For example, details are described in Japanese Patent No. 5,354,216, and this technology can also be used in the present invention. In other words, when the probe hybridizes, fluorescence can be quenched if the guanine base in the amplified product is located within a range of, for example, 1 to 3 bases of the cytosine base in the probe (a base that forms a base pair with a cytosine base is considered to be 1).

In a further specific embodiment, even if at least one terminal base labeled with a fluorescence 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. 5,354,216, and this technology can also be used in the present invention. For example, when the probe hybridizes, quenching can be achieved if a guanine base in the amplification product is located within, for example, 1 to 3 bases of at least one terminal base labeled with a fluorescence 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 mode 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 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 in real time.
(2-2b) After completion of the step (2-1), the nucleic acid probe is hybridized to the amplification product that can be obtained, and the fluorescence intensity of the reaction solution is measured to monitor the progress of the nucleic acid amplification reaction at an endpoint.
(2-2c) After completion of the step (2-1), the nucleic acid probe is hybridized to the amplification product that can be obtained, and the temperature dependence of the fluorescence intensity of the reaction solution is measured.
According to the steps (2-2a) to (2-2c) described above, it may be possible to detect the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe simply and with high sensitivity.

(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 furthermore, the target nucleic acid contained in the sample can be quantified based on the amplification rate.
For example, the progress of a nucleic acid amplification reaction can be monitored in real time by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescence 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 Bordetella pertussis or Bordetella parapertussis, 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) Monitoring the progress of nucleic acid amplification reactions at endpoints
By monitoring the progress of the nucleic acid amplification reaction at the endpoint, the target nucleic acid contained in the sample can be rapidly detected. Furthermore, by comparing the fluorescence intensity at the endpoint, the target nucleic acid can be quantified.
For example, the progress of a nucleic acid amplification reaction is monitored at an endpoint by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescence quenching dye (see, for example, Example 3). 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 simplifying the detection step, it is preferable to monitor it at an end point.

[(2-2c) Measurement of temperature dependence of fluorescence intensity]
Specifically, measuring the temperature dependence of fluorescence intensity involves measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high (see, for example, Example 4). The melting temperature (Tm value) specific to the oligonucleotide probe used can be determined by first differentiating the obtained fluorescence intensity with respect to temperature. Furthermore, the fluorescence intensity may be converted into a fluorescence quenching rate or the like depending on the purpose.
The detection, analysis, etc. of a target nucleic acid using the Tm value is called melting curve analysis. Generally, 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 remains 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 in a target nucleic acid. The base sequence polymorphism referred to here includes single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

For example, melting curve analysis is also applied to SNP analysis.
When the base sequence of the target nucleic acid is mutated relative to the oligonucleotide probe, the bases are mismatched when the probe hybridizes, generally resulting in a low Tm value. Therefore, single nucleotide polymorphism analysis (SNP analysis) can be performed by comparing the magnitude of the Tm values.

[Specimen sample]
The specimen sample that can be used in the present invention is not particularly limited as long as it may contain Bordetella bacteria. Examples include, but are not limited to, oral scrapings, throat swabs, nasal swabs, nasal aspirates, sputum, bronchial lavage fluid, and alveolar lavage fluid collected from subjects suspected of infection with B. pertussis or B. parapertussis. When a biological sample is used as the specimen sample to be measured, pretreatment such as dilution, suspension, or centrifugation, or nucleic acid extraction may be performed depending on the biological sample, although this is not particularly limited.

There are no particular limitations on the collection and preparation methods for specimen samples, and known methods can be used depending on the type of sample and purpose. In a particularly preferred embodiment, the specimen sample used in the present invention can be a sample obtained by alkaline treatment (e.g., pH 8.0 to 14.0, preferably pH 10.0 to 13.0) of a specimen collected from a living body.

The method for nucleic acid extraction is not particularly limited, and known methods can be used depending on the type of sample and purpose. For example, kits sold by various manufacturers may be used for nucleic acid extraction.

In certain preferred embodiments, the method of the present invention does not require a nucleic acid purification step, which is typically considered essential in conventional nucleic acid amplification reactions. As shown in the test examples described below, the method of the present invention has been confirmed to enable highly sensitive detection of Bordetella spp. even in specimens containing contaminants other than the target nucleic acid, such as biologically derived components (which may inhibit nucleic acid amplification reactions). Nucleic acid purification requires specialized reagents and is cumbersome, time-consuming, and laborious. However, the gene amplification reaction of the present invention eliminates the need for specialized equipment for nucleic acid purification, eliminating this tedious process. Furthermore, the time required for centrifugation can be reduced, thereby shortening the time from sample collection to obtaining genetic test results. For example, the time from sample collection to obtaining genetic test results can be reduced to 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 (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, eliminates the need for nucleic acid purification and is therefore preferable as it enables Bordetella bacteria to be detected simply and quickly.

[Bordetella spp.]
The genus Bordetella is a gram-negative coccobacillus known to be a causative agent of various respiratory diseases. Bordetella bacteria are known to cause various infectious diseases, such as B. pertussis, which causes Bordetella pertussis, B. parapertussis, which causes Bordetella, and B. bronchiseptica, which causes bronchitis. According to the method of the present invention, any of these Bordetella bacteria can be detected with high sensitivity. Preferably, the method of the present invention can accurately detect and distinguish between B. pertussis and B. parapertussis.

[Reagents for distinguishing and detecting Bordetella species]
Another embodiment of the present invention is a reagent for distinguishing and detecting Bordetella bacteria that may be present 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 vary depending on the nucleic acid amplification reaction to be performed, and known methods can be used for each. For example, when detecting Bordetella bacteria that may be present in a sample using a PCR reaction, the reagent preferably contains at least a DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and an inorganic salt such as a magnesium salt. The concentration of each component can be adjusted appropriately depending on the intended experiment. 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 to suppress nonspecific amplification or promote the reaction. Additives for suppressing nonspecific amplification include known anti-DNA polymerase antibodies and phosphate. 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 the present invention is not limited to these.

[Kit for distinguishing and detecting Bordetella genus bacteria]
Another embodiment of the present invention is a kit for differentially detecting Bordetella bacteria that may be contained in a sample. The kit of the present invention is not particularly limited as long as it includes the nucleic acid primer set, nucleic acid probe, and/or reagents containing these of the present invention described above and is configured to differentially detect Bordetella bacteria. For example, the kit of the present invention can optionally include a genetic testing reagent capable of detecting or quantifying the presence of the target substance and/or instructions for use, etc. For example, the nucleic acid primer set and nucleic acid probe can be enclosed in the same container or in separate containers, and packaged in a single package, for example, and provided in a form that includes information on how to use the kit.

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

Example 1: Detection of Bordetella pertussis
(1) Sample Preparation DNA samples extracted from B. pertussis or B. parapertussis 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 B. pertussis or B. parapertussis 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, manufactured by Toyobo) 3 μL
PPD Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
IC Mix (GeneCube® Test Basic, manufactured by 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: 16 0.25 μL
3 μL of sample

Nucleic acid amplification and melting curve analysis: 94°C, 2 minutes (one cycle)
97℃ 1 second 58℃ 3 seconds 63℃ 5 seconds (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 changes 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 fluorescence signal. In the graph, BP genome represents the analytical results of B. pertussis genomic DNA, BPP genome represents the analytical results of B. parapertussis genomic DNA samples, and NC represents the analytical results of sterilized water. As is clear from Figure 1, B. pertussis and B. parapertussis can be detected distinctly by the present invention.

Example 2: Detection of Bordetella pertussis
(1) Sample Preparation A DNA sample (BP genome) extracted from Bordetella pertussis was suspended in saline and mixed with a postnasal or pharyngeal 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 Bordetella pertussis 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, manufactured by Toyobo) 3 μL
PPD Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
IC Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
10 μM Primer shown in SEQ ID NO: 12 0.25 μL
10 μM Primer shown in SEQ ID NO: 3 1.5 μL
10 μM Probe shown in SEQ ID NO: 17 0.25 μL
3 μL of sample

Nucleic acid amplification and melting curve analysis: 94°C, 2 minutes (one cycle)
97°C for 1 second, 60°C for 3 seconds, 63°C for 6 seconds (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 2 shows the results of nucleic acid amplification and detection performed under the above conditions. The results are shown for postnasal swabs and pharyngeal swabs to which Bordetella pertussis genomic DNA was added. As shown in Figure 2, Bordetella pertussis was detected in postnasal swabs and pharyngeal swabs. These results revealed, quite unexpectedly, that Bordetella pertussis could be detected without being affected by amplification inhibition contained in the specimen.

Example 3: Detection of Bordetella pertussis
(1) Sample Preparation A DNA sample (BP genome) extracted from Bordetella pertussis was suspended in saline and mixed with a postnasal or pharyngeal 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 Bordetella pertussis 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, manufactured by Toyobo) 3 μL
PPD Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
IC Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
10 μM Primer shown in SEQ ID NO: 8 0.25 μL
10 μM Primer shown in SEQ ID NO: 15 1.5 μL
10 μM Probe shown in SEQ ID NO: 19 0.25 μL
3 μL of sample

Nucleic acid amplification and melting curve analysis: 94°C, 2 minutes (one cycle)
97°C for 1 second, 60°C for 3 seconds, 63°C for 6 seconds (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 3 shows the results of nucleic acid amplification and detection performed under the above conditions. The figures show the measurement results of postnasal swabs or pharyngeal swabs to which Bordetella pertussis genomic DNA was added. As shown in Figure 3, pertussis was also detected in postnasal swabs and pharyngeal swabs. These specimens are known to contain nucleic acid amplification inhibitors such as viscous substances (e.g., mucin) derived from living organisms. The results of this example demonstrate that Bordetella pertussis can be detected without being affected by amplification inhibition contained in such specimens.

Example 4: Detection of B. parapertussis
(1) Sample Preparation: Throat swabs were suspended in eSwab medium (manufactured by Copan), and a DNA sample (BPP genome) extracted from B. parapertussis was mixed with the suspended throat swabs in eSwab medium to prepare a pseudo-biological sample at 2 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. Additionally, a DNA sample (BPP genome) extracted from B. parapertussis was prepared in sterile water at 2 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 Example Reagent, respectively, and B. parapertussis 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, manufactured by Toyobo) 3 μL
PPD Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
IC Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
10 μM Primer shown in SEQ ID NO: 6 0.25 μL
10 μM Primer shown in SEQ ID NO: 10 1.5 μL
10 μM Probe shown in SEQ ID NO: 18 0.25 μL
3 μL of sample

Furthermore, as a comparative reagent, a reagent that was essentially the same as that of the present invention was prepared, except that the following nucleic acid primers and nucleic acid probes (SEQ ID NOs: 21 to 23: nucleic acid primers and nucleic acid probes described in Patent Document 3) different from those of the present invention were used, and this was used in a comparison experiment.
10 μM Primer shown in SEQ ID NO: 21 0.25 μL
10 μM Probe represented by SEQ ID NO: 22 0.25 μL
10 μM Primer shown in SEQ ID NO: 23 1.5 μL

Nucleic acid amplification and melting curve analysis: 94°C, 2 minutes (one cycle)
97°C 1 second, 58°C 3 seconds, 63°C 6 seconds (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. Both the reagent of this example and the reagent of the comparative example detected Bordetella parapertussis in the positive samples. On the other hand, the reagent of the comparative example was unable to detect Bordetella parapertussis in the simulated biological sample due to amplification inhibition caused by substances contained in the specimen, whereas the present reagent was free from inhibition and enabled highly sensitive detection. The results of this example demonstrate that the use of the nucleic acid primers and nucleic acid probes of the present invention can suppress the influence of contaminants that may inhibit the amplification reaction, even in biological samples that have not undergone a nucleic acid purification process, thereby enabling highly sensitive detection of Bordetella spp.

Example 5: Detection of Bordetella pertussis and Bordetella parapertussis
(1) Sample Preparation DNA samples extracted from Bordetella pertussis and Bordetella parapertussis were adjusted with sodium hydroxide solution to 0.5 (copies/μL), 0.25 (copies/μL), 0.15 (copies/μL), and 0.075 (copies/μL), respectively, to prepare samples (pH 12.4 after adjustment).
(2) Nucleic Acid Amplification and Melting Curve Analysis The above samples were added to the following reagents, and Bordetella pertussis and Bordetella parapertussis were measured under the following conditions (n = 4). 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, manufactured by Toyobo) 4 μL
PPD Mix (GeneCube® Test Basic, manufactured by Toyobo) 1.9 μL
IC Mix (GeneCube® Test Basic, manufactured by Toyobo) 1.3 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 shown in SEQ ID NO: 18 0.25 μL
4 μL of sample

Nucleic acid amplification and melting curve analysis: 94°C, 2 minutes (one cycle)
97°C 1 second, 58°C 3 seconds, 63°C 6 seconds (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 Table 1 below summarizes the results of nucleic acid amplification detection under the above conditions, where low concentrations of Bordetella pertussis and Bordetella parapertussis were detected. The numbers detected are shown for n = 4 measurements. Table 1 shows that Bordetella pertussis could be detected down to 0.25 (copies/μL) and Bordetella parapertussis down to 0.15 (copies/μL), demonstrating that high sensitivity is possible even with low copy numbers.

Example 6: Detection of Bordetella pertussis and Bordetella parapertussis
(1) Sample Preparation Postnasal swabs and pharyngeal swabs were suspended in water and subjected to Bordetella detection by another method (real-time PCR). The following tests were performed using samples that were confirmed to be positive for Bordetella pertussis or Bordetella parapertussis. These samples were used as they were without further nucleic acid extraction or purification.
(2) Nucleic Acid Amplification and Melting Curve Analysis The above samples were added to the following reagents, and Bordetella pertussis and Bordetella parapertussis were measured 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, manufactured by Toyobo) 3 μL
PPD Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
IC Mix (GeneCube® Test Basic, manufactured by Toyobo) 1 μL
10 μM Primer shown in SEQ ID NO: 4 0.25 μL
10 μM Primer shown in SEQ ID NO: 10 1.5 μL
10 μM Probe shown in SEQ ID NO: 18 0.25 μL
3 μL of sample

Nucleic acid amplification and melting curve analysis: 94°C, 2 minutes (one cycle)
97°C 1 second, 58°C 3 seconds, 63°C 6 seconds (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 5 shows the results of nucleic acid amplification and detection performed under the above conditions. It can be seen that this reagent was able to detect and distinguish between Bordetella pertussis and Bordetella parapertussis with sufficient sensitivity, even in actual specimens (positive specimens) containing biological contaminants.

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 Bordetella bacteria that may be present in a sample. Therefore, for example, the present invention makes it possible to accurately distinguish and detect B. pertussis and B. parapertussis, which have previously been difficult to distinguish, using a simple method, and can make a significant contribution to clinical diagnosis, etc.

Claims (12)

A nucleic acid probe used to distinguish and detect B. pertussis and B. parapertussis that may be contained in a specimen sample, the nucleic acid probe having the following characteristics (A) and (B):
(A) consisting of a base sequence shown in any one of SEQ ID NOs: 16 to 20, or any of these base sequences in which one base has been substituted, deleted, or added ; and (B) at least one terminal base is labeled with a fluorescence quenching dye that quenches fluorescence by interaction with guanine. 2. The nucleic acid probe according to claim 1 , 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. 3. The nucleic acid probe according to claim 1, 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). 4. The nucleic acid probe according to claim 1 , wherein in (B), at least one terminal base labeled with a fluorescence quenching dye is cytosine.
A method for distinguishing and detecting B. pertussis and B. parapertussis that may be contained in a specimen sample, the method comprising the steps of:
(1) performing a nucleic acid amplification reaction in a specimen sample using a nucleic acid primer set including a nucleic acid primer corresponding to the following (I) and a nucleic acid primer corresponding to the following (II) to generate a nucleic acid amplification product;
(2-1) hybridizing the nucleic acid amplification product obtained in the step (1) with the nucleic acid probe according to any one of claims 1 to 4 to form a complex; and (2-2) detecting the complex obtained in the step (2-1):
(I) a nucleic acid primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 3 to 9, or a nucleotide sequence in which one nucleotide has been substituted, deleted, or added ;
(II) A nucleic acid primer consisting of a base sequence shown in any one of SEQ ID NOs: 10 to 15, or a base sequence in which one base has been substituted, deleted, or added in such a base sequence . The detection method according to claim 5 , 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 that contains or does not contain a complex formed by hybridizing the nucleic acid probe to the nucleic acid amplification product.
(2-2b) A step of monitoring the progress of the nucleic acid amplification reaction at an endpoint by measuring the fluorescence intensity of a reaction solution that 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 probe to the nucleic acid amplification product. The detection method according to claim 5 or 6 , wherein the nucleic acid amplification reaction in step (1) is PCR. 8. The detection method according to claim 5 , 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. The detection method according to claim 8 , 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 5 to 9 , wherein the specimen sample is a biological sample that has not been subjected to a nucleic acid purification step. A reagent for distinguishing between and detecting B. pertussis and B. parapertussis, comprising a nucleic acid primer set including the nucleic acid probe according to any one of claims 1 to 4 and a nucleic acid primer corresponding to the following (I) and (II):
(I) a nucleic acid primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 3 to 9, or a nucleotide sequence in which one nucleotide has been substituted, deleted, or added ;
(II) A nucleic acid primer consisting of a base sequence shown in any one of SEQ ID NOs: 10 to 15, or a base sequence in which one base has been substituted, deleted, or added in such a base sequence . The nucleic acid probe according to any one of claims 1 to 4 .
A kit for distinguishing between and detecting B. pertussis and B. parapertussis, comprising: a nucleic acid primer set including a nucleic acid primer corresponding to the following (I) and a nucleic acid primer corresponding to the following (II); and /or the reagent according to claim 11 :
(I) a nucleic acid primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 3 to 9, or a nucleotide sequence in which one nucleotide has been substituted, deleted, or added ;
(II) A nucleic acid primer consisting of a base sequence shown in any one of SEQ ID NOs: 10 to 15, or a base sequence in which one base has been substituted, deleted, or added in such a base sequence .