Patent
Disclosed herein are methods and compositions for detecting Bordetella pertussis and Bordetella parapertussis by detecting the presence of the IS481 and IS1001 genomic insertion sequences, respectively.
1. A method for identifying the presence or absence of Bordetella pertussis and Bordetella parapertussis in a biological sample, comprising:
(a) providing a first primer pair suitable for amplifying an IS481 target nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 or a full complement thereof, amplifying said IS481 target sequence if present in the sample, and detecting the IS481 target sequence by hybridizing it with an oligonucleotide probe, wherein one primer of the first primer pair comprises a 3′-primer element and a 5′-probe tail, wherein said 5′-probe tail comprises a hairpin structure comprising the oligonucleotide probe and a fluorophore/quencher pair; and
(b) providing a second primer pair suitable for amplifying an IS1001 target nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9 or a full complement thereof, amplifying said IS1001 target sequence if present in the sample, and detecting the IS1001 target sequence by hybridizing with an oligonucleotide probe, wherein one primer of the second primer pair comprises a 3′-primer element, and a 5′-probe tail, wherein said 5′-probe tail comprises a hairpin structure comprising the oligonucleotide probe and a fluorophore/quencher pair, and comprises the sequence of SEQ ID NOS: 14 and 31 in 5′-to-3′ orientation,
wherein the presence of said IS481 target nucleic acid identifies the presence of B. pertussis , and the presence of said IS1001 target nucleic acid identifies the presence of B. parapertussis.
2. The method of claim 1 , wherein one primer of the first primer pair comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 25.
3. The method of claim 1 , wherein one primer of the first primer pair comprises the sequence of (a) SEQ ID NOS: 13, 29, and 30 in 5′-to-3′ orientation or (b) SEQ ID NOS: 28 and 30 in 5′-to-3′ orientation.
4. The method of claim 1 , wherein one primer of the second primer pair comprises the sequence of SEQ ID NO: 26.
5. The method of claim 1 , wherein said method comprises real-time PCR.
6. The method of claim 1 wherein said amplifying in step a) and said amplifying in step b) are conducted simultaneously in a single reaction.
7. The method of claim 1 , wherein the 5′-probe tail and the 3′-primer element of the first and second primer pairs are connected by an amplification blocker.
8. A method for identifying the presence or absence of Bordetella pertussis and Bordetella parapertussis in a biological sample, comprising:
(a) providing a first primer pair suitable for amplifying an IS481 target nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 or a full complement thereof, amplifying said IS481 target sequence if present in the sample, and detecting the IS481 target sequence by hybridizing it with an oligonucleotide probe, wherein one primer of the first primer pair comprises a 3′-primer element and a 5′-probe tail, wherein said 5′-probe tail comprises a hairpin structure comprising the oligonucleotide probe and a fluorophore/quencher pair, and comprises the sequence of (a) SEQ ID NOS: 13, 29, and 30 in 5′-to-3′ orientation or (b) SEQ ID NOS: 28 and 30 in 5′- to -3′ orientation; and
(b) providing a second primer pair suitable for amplifying an IS1001 target nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9 or a full complement thereof, amplifying said IS1001 target sequence if present in the sample, and detecting the IS1001 target sequence by hybridizing with an oligonucleotide probe, wherein one primer of the second primer pair comprises a 3′-primer element and a 5′-probe tail, wherein said 5′-probe tail comprises a hairpin structure comprising the oligonucleotide probe and a fluorophore/quencher pair; and
wherein the presence of said IS481 target nucleic acid identifies the presence of B. pertussis , and the presence of said IS1001 target nucleic acid identifies the presence of B. parapertussis.
9. The method of claim 8 , wherein said amplifying in step a) and said amplifying in step b) are conducted simultaneously in a single reaction.
10. The method of claim 8 , wherein the 5′-probe tail and the 3′-primer element of the first and second primer pairs are connected by an amplification blocker.
11. The method of claim 8 , wherein one primer of the first primer pair comprises the sequence of SEQ ID NO: 25.
12. The method of claim 8 , wherein one primer of the second primer pair comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 26.
13. The method of claim 12 , wherein one primer of the second primer pair comprises the sequence of SEQ ID NOS: 14 and 31 in 5′-to-3′ orientation.