Probes for detecting Bordetella pertussis and their applications
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- TOYOBO CO LTD
- Filing Date
- 2025-11-13
- Publication Date
- 2026-06-23
AI Technical Summary
【0010】 本発明により、百日咳菌の検出に有用なプローブ及びそれを用いた百日咳菌の検出方法等を提供することができる。当該検出方法は、例えば、百日咳菌を簡便、迅速、且つ高感度に検出することができる。また、本発明では、1種類のプローブを用いることにより、野生型及び抗生物質耐性を示す変異型の両方の百日咳菌を検出することも可能である。さらに、本発明では、百日咳菌を野生型及び抗生物質耐性を示す変異型のいずれであるかを判別して検出することも可能である。本発明のプローブ、それを用いた検出方法、試薬、又はキット等を使用することで、百日咳菌の簡便、迅速、且つ高感度な検出が可能になり、臨床診断の分野に大きく貢献できる。
Smart Images
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Abstract
Claims
1. A probe for detecting Bordetella pertussis having the following characteristics (A) and (B): (A) Containing the following base sequence (A-1) or (A-2); (A-1) A nucleotide sequence including at least the 92nd base in the region of the nucleotide sequence shown in Sequence ID No. 1, or a nucleotide sequence of at least 12 consecutive bases in a nucleotide sequence complementary to the said nucleotide sequence, (A-2) A base sequence in which 1 to 3 bases are substituted, deleted, inserted, or added in the base sequence of (A-1); and (B) Only the 5' end or the 3' end is labeled.
2. The probe according to claim 1, wherein the base sequence of (A) includes a corresponding base at the position corresponding to position 2047 of the Bordetella pertussis 23S rRNA gene sequence, and the corresponding base at the position corresponding to position 2047 of the Bordetella pertussis 23S rRNA gene sequence is an adenine base or a guanine base.
3. The probe according to claim 1, wherein the length of the base sequence of (A) is 12 to 30 bases.
4. The probe according to claim 1, wherein the base sequence of (A) includes the base sequence shown in any of SEQ ID NOs: 2 to 10 and 15 to 22 or a base sequence complementary thereto.
5. The probe according to claim 1, wherein the label in (B) is a fluorescent dye label.
6. The probe according to claim 1, wherein the label (B) is a fluorescent quenching dye that quenches when bound to a nucleic acid containing a base sequence that exhibits 90% or more identity with a base sequence complementary to the base sequence of the probe.
7. The probe according to claim 1, wherein the label (B) is a fluorescent quenching dye that is quenched by interaction with guanine.
8. The probe according to claim 1, wherein the label (B) is a label with at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.
9. The probe according to claim 1, wherein the label (B) is labeled with at least one fluorescent quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).
10. The probe according to claim 1, wherein the labeled terminal base in (B) is cytosine.
11. A method for detecting Bordetella pertussis that may be present in a sample using a probe according to any one of claims 1 to 10.
12. The following steps (1), (2), and (3): (1) A step of providing a sample that may contain Bordetella pertussis, (2) A step of subjecting the sample from step (1) to a nucleic acid amplification reaction using the region containing position 2047 of the Bordetella pertussis 23S rRNA gene sequence as a template, and (3) A step of detecting one or more nucleic acid amplification products that can be produced in the nucleic acid amplification reaction of step (2) using one or more of the probes, The method according to claim 11, including the method described in claim 11.
13. The method according to claim 12, wherein step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.
14. The method according to claim 13, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a variant thereof.
15. The method according to claim 12, wherein the primer set used in the nucleic acid amplification reaction of step (2) is a primer set comprising a combination of a first primer having a nucleotide sequence S13 of 15 to 35 bases consecutively in nucleotide sequence S11 at nucleotide sequences 1 to 80 of SEQ ID NO: 1 or nucleotide sequence S12 complementary to nucleotide sequence S11, or a nucleotide sequence S14 in which 1 to 3 bases are substituted, deleted, inserted, or added in nucleotide sequence S13, and a second primer having a nucleotide sequence S24 in which 15 to 35 bases consecutively in nucleotide sequences S21 at nucleotide sequences 105 to 151 of SEQ ID NO: 1 or nucleotide sequence S22 complementary to nucleotide sequence S21, or a nucleotide sequence S24 in which 1 to 3 bases are substituted, deleted, inserted, or added in nucleotide sequence S23, wherein the second primer is complementary to the DNA elongation product of the first primer.
16. The method according to claim 15, wherein the first primer has a base sequence S15 represented by any one of SEQ ID NOs: 11, 12, and 23 or a complementary base sequence S16 thereto, or a base sequence S17 obtained by substituting, deleting, inserting, or adding 1 to 3 bases in base sequence S15 or S16, and the second primer has a base sequence S25 represented by any one of SEQ ID NOs: 13, 14, and 24 or a complementary base sequence S26 thereto, or a base sequence S27 obtained by substituting, deleting, inserting, or adding 1 to 3 bases in base sequence S25 or S26.
17. The method according to claim 11, for detecting both the wild type and antibiotic-resistant variant of Bordetella pertussis.
18. The method according to claim 11, for detecting Bordetella pertussis by determining whether it is wild-type or antibiotic-resistant mutant.
19. The method according to claim 12, wherein the detection step in step (3) is performed by melting curve analysis.
20. The method according to claim 19, wherein, in the melting curve analysis, the detection temperature of the antibiotic-resistant mutant is detected at a lower temperature than the detection temperature of the wild type.
21. The method according to claim 19, wherein, in the melting curve analysis, the difference between the detection temperature of the antibiotic-resistant mutant and the detection temperature of the wild type is 5°C or more.
22. A reagent or kit for detecting Bordetella pertussis, comprising the probe described in any one of claims 1 to 10.
23. A primer set comprising a combination of a first primer having a nucleotide sequence S13 of 15 to 35 bases consecutively in the nucleotide sequence S11 at positions 1 to 80 of the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence S12 complementary to nucleotide sequence S11, or a nucleotide sequence S14 obtained by substituting, deleting, inserting, or adding 1 to 3 bases in nucleotide sequence S13, and a second primer having a nucleotide sequence S24 obtained by substituting, deleting, inserting, or adding 15 to 35 bases consecutively in the nucleotide sequence S21 at positions 105 to 151 of the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence S22 complementary to nucleotide sequence S21, wherein the second primer is complementary to the DNA elongation product of the first primer.