Compositions and methods for detecting
Disclosed are nucleic acid oligomers, including amplification oligomers and detection probes, for detection of Bordetella pertussis and Bordetella parapertussis nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.
1. A composition for determining the presence or absence of each of Bordetella pertussis (Bp) and Bordetella parapertussis (Bpp) in a sample, said composition comprising:
a first amplification oligomer combination and a second amplification oligomer combination, wherein
(I) the first amplification oligomer combination comprises first and second Bp-specific amplification oligomers capable of amplifying a target region of a Bp target nucleic acid, wherein the first and second Bp-specific amplification oligomers comprise, respectively, a first Bp-specific target-hybridizing sequence and a second Bp-specific target-hybridizing sequence, wherein
(a) the first Bp-specific target hybridizing sequence is SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:45, a sequence having at least 85% identity with SEQ ID NO: 3, a sequence having at least 85% identity with SEQ ID NO:6, or an RNA equivalent or DNA/RNA chimeric thereof, and
the second Bp-specific target-hybridizing sequence is SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO:11, or an RNA equivalent or DNA/RNA chimeric thereof; or
(b) the first Bp-specific target-hybridizing sequence is SEQ ID NO:18, SEQ ID NO: 21, or SEQ ID NO:27, or an RNA equivalent or DNA/RNA chimeric thereof, and
the second Bp-specific target-hybridizing sequence is SEQ ID NO:20 or SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof;
and
(II) the second amplification oligomer combination comprises first and second Bpp-specific amplification oligomers capable of amplifying a target region of a Bpp target nucleic acid, wherein the first and second Bpp-specific amplification oligomers comprise, respectively, first and second Bpp-specific target-hybridizing sequences, wherein
(a) the first Bpp-specific target-hybridizing sequence is SEQ ID NO:30, SEQ ID NO: 36, or SEQ ID NO:39, or an RNA equivalent or DNA/RNA chimeric thereof, and
the second Bpp-specific target-hybridizing sequence is SEQ ID NO:32, SEQ ID NO: 38, or SEQ ID NO:41, or an RNA equivalent or DNA/RNA chimeric thereof;
(b) the first Bpp-specific target-hybridizing sequence is SEQ ID NO:33, or an RNA equivalent or DNA/RNA chimeric thereof, and
the second Bpp-specific target-hybridizing sequence is SEQ ID NO:35, or an RNA equivalent or DNA/RNA chimeric thereof; or
(c) the first Bpp-specific target-hybridizing sequence is SEQ ID NO:42, or an RNA equivalent or DNA/RNA chimeric thereof, and
the second Bpp-specific target-hybridizing sequence is SEQ ID NO:44, or an RNA equivalent or DNA/RNA chimeric thereof; and
at least one detection probe oligomer configured to specifically hybridize to a Bp or Bpp target sequence that is amplifiable using the first and second Bp-specific amplification oligomers or the first and second Bpp-specific amplification oligomers, wherein the at least one detection probe oligomer comprises a fluorescent or chemiluminescent label.
2. The composition of claim 1 , wherein the first and second Bp-specific target-hybridizing sequences are, respectively, selected from the group consisting of
(i) SEQ ID NO:3, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:5, or an RNA equivalent or DNA/RNA chimeric thereof;
(ii) SEQ ID NO:6, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:8, or an RNA equivalent or DNA/RNA chimeric thereof;
(iii) SEQ ID NO:9, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:11, or an RNA equivalent or DNA/RNA chimeric thereof;
(iv) SEQ ID NO:18, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:20, or an RNA equivalent or DNA/RNA chimeric thereof; and
(v) SEQ ID NO:21, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:23, or an RNA equivalent or DNA/RNA chimeric thereof.
3. The composition of claim 2 , wherein the at least one detection probe oligomer comprises a Bp-specific detection probe oligomer comprising a Bp-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a Bp amplicon amplifiable by the first and second Bp-specific amplification oligomers; optionally wherein
the first and second Bp-specific target-hybridizing sequences are target-hybridizing sequences of (i) and the Bp-specific detection probe target-hybridizing sequence is a sequence having at least 85% identity with SEQ ID NO:4, or an RNA equivalent or DNA/RNA chimeric thereof;
the first and second Bp-specific target-hybridizing sequences are target-hybridizing sequences of (ii) and the Bp-specific detection probe target-hybridizing sequence is a sequence having at least 85% identity with SEQ ID NO:7, or an RNA equivalent or DNA/RNA chimeric thereof;
the first and second Bp-specific target-hybridizing sequences are target-hybridizing sequences of (iii) and the Bp-specific detection probe target-hybridizing sequence is SEQ ID NO:10, or an RNA equivalent or DNA/RNA chimeric thereof;
the first and second Bp-specific target-hybridizing sequences are target-hybridizing sequences of (iv) and the Bp-specific detection probe target-hybridizing sequence is SEQ ID NO: 16, or an RNA equivalent or DNA/RNA chimeric thereof; or
the first and second Bp-specific target-hybridizing sequences are target-hybridizing sequences of (v) and the Bp-specific detection probe target-hybridizing sequence is SEQ ID NO: 19, or an RNA equivalent or DNA/RNA chimeric thereof.
4. The composition of claim 1 , wherein the first and second Bpp-specific target-hybridizing sequences are, respectively, selected from the group consisting of
(i′) SEQ ID NO:30, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:32, or an RNA equivalent or DNA/RNA chimeric thereof;
(ii′) SEQ ID NO:33, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:35, or an RNA equivalent or DNA/RNA chimeric thereof;
(iii′) SEQ ID NO:36, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:38, or an RNA equivalent or DNA/RNA chimeric thereof;
(iv′) SEQ ID NO:39, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:41, or an RNA equivalent or DNA/RNA chimeric thereof; and
(v′) SEQ ID NO:42, or an RNA equivalent or DNA/RNA chimeric thereof, and SEQ ID NO:44, or an RNA equivalent or DNA/RNA chimeric thereof.
5. The composition of claim 4 , wherein the at least one detection probe oligomer comprises a Bpp-specific detection probe oligomer comprising a Bpp-specific detection probe target-hybridizing sequence that is from about 15 to about 35 nucleotides in length and is configured to hybridize to a target sequence contained within a Bpp amplicon amplifiable by the first and second Bpp-specific amplification oligomers; optionally wherein
the first and second Bpp-specific target-hybridizing sequences are target-hybridizing sequences of (i′) and the Bpp-specific detection probe target-hybridizing sequence is SEQ ID NO:31, or an RNA equivalent or DNA/RNA chimeric thereof;
the first and second Bpp-specific target-hybridizing sequences are target-hybridizing sequences of (ii′) and the Bpp-specific detection probe target-hybridizing sequence is SEQ ID NO:34, or an RNA equivalent or DNA/RNA chimeric thereof;
the first and second Bpp-specific target-hybridizing sequences are target-hybridizing sequences of (iii′) and the Bpp-specific detection probe target-hybridizing sequence is SEQ ID NO:37, or an RNA equivalent or DNA/RNA chimeric thereof;
the first and second Bpp-specific target-hybridizing sequences are target-hybridizing sequences of (iv′) and the Bpp-specific detection probe target-hybridizing sequence is SEQ ID NO:40, or an RNA equivalent or DNA/RNA chimeric thereof; or
the first and second Bpp-specific target-hybridizing sequences are target-hybridizing sequences of (v′) and the Bpp-specific detection probe target-hybridizing sequence is SEQ ID NO:43, or an RNA equivalent or DNA/RNA chimeric thereof.
6. A kit for determining the presence or absence of each of Bordetella pertussis (Bp) and Bordetella parapertussis (Bpp) in a sample, said kit comprising the composition of claim 1 .
7. A method for determining the presence or absence of each of Bordetella pertussis (Bp) and Bordetella parapertussis (Bpp) in a sample, said method comprising:
(1) contacting a sample suspected of containing at least one of Bordetella pertussis (Bp) and Bordetella parapertussis (Bpp) with the first amplification oligomer combination and the second amplification oligomer combination according to claim 1 ;
(2) performing an in vitro nucleic acid amplification reaction, wherein any Bp target nucleic acid, if present in the sample, is used as a template for generating one or more amplicons corresponding to the Bp target region, and wherein any Bpp target nucleic acid, if present in the sample, is used as a template for generating one or more amplicons corresponding to the Bpp target region; and
(3) detecting the presence or absence of the one or more amplicons, thereby determining the presence or absence of Bp and Bpp in the sample.
8. The composition of claim 1 , wherein the first Bp-specific target-hybridizing sequence having at least 85% identity with SEQ ID NO:3 is SEQ ID NO:3 or SEQ ID NO:6, or an RNA equivalent or DNA/RNA chimeric thereof.
9. The composition of claim 3 , wherein the Bp-specific detection probe target-hybridizing sequence having at least 85% identity with SEQ ID NO:4 is SEQ ID NO:4 or SEQ ID NO:7, or an RNA equivalent or DNA/RNA chimeric thereof.
10. The method of claim 7 , wherein the first Bp-specific target-hybridizing sequence having at least 85% identity with SEQ ID NO:3 is SEQ ID NO:3 or SEQ ID NO:6, or an RNA equivalent or DNA/RNA chimeric thereof.
11. The composition of claim 1 , wherein the first and second Bp-specific amplification oligomers comprise the first and second Bp-specific target-hybridizing sequences of (I)(a).
12. The composition of claim 11 , wherein the first Bp-specific target-hybridizing sequence is the sequence having at least 85% identity with SEQ ID NO:3, a sequence having at least 85% identity with SEQ ID NO:6, or an RNA equivalent or DNA/RNA chimeric thereof.
13. The composition of claim 12 , wherein the second Bp-specific target-hybridizing sequence is SEQ ID NO:5 or SEQ ID NO:8, or an RNA equivalent or DNA/RNA chimeric thereof.
14. The composition of claim 13 , wherein the at least one detection probe oligomer comprises a Bp-specific detection probe oligomer comprising a Bp-specific detection probe target-hybridizing sequence that is a sequence having at least 85% identity with SEQ ID NO: 4, a sequence having at least 85% identity with SEQ ID NO:7, or an RNA equivalent or DNA/RNA chimeric thereof.
15. The composition of claim 1 , wherein the first and second Bpp-specific amplification oligomers comprise the first and second Bpp-specific target-hybridizing sequences of (II)(a).
16. The composition of claim 15 , wherein the first Bpp-specific target-hybridizing sequence is SEQ ID NO:30, or an RNA equivalent or DNA/RNA chimeric thereof.
17. The composition of claim 16 , wherein the second Bpp-specific target-hybridizing sequence is SEQ ID NO:32, or an RNA equivalent or DNA/RNA chimeric thereof.
18. The composition of claim 17 , wherein the at least one detection probe oligomer comprises a Bpp-specific detection probe oligomer comprising a Bpp-specific detection probe target-hybridizing sequence that is SEQ ID NO:31, or an RNA equivalent or DNA/RNA chimeric thereof.
19. The composition of claim 13 , wherein the first Bpp-specific target-hybridizing sequence is SEQ ID NO:30, or an RNA equivalent or DNA/RNA chimeric thereof.
20. The composition of claim 19 , wherein the second Bpp-specific target-hybridizing sequence is SEQ ID NO:32, or an RNA equivalent or DNA/RNA chimeric thereof.