Amplification and detection of mycobacterium avium complex species
AssigneeBecton, Dickinson and Company
InventorsKaren Ann Dilly, Silvia A. Bustos, Christine Ann Rostkowski, Dolores M. Berger
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Abstract
Amplification primers and methods for complex-specific amplification of target sequences in the dnaJ genes of the Mycobacterium avium Complex (MAC) species are disclosed. The primer target binding sequences are useful for amplification of the dnaJ target in a variety of amplification reactions, with detection of the complex-specific target and, optionally, identification of the MAC species from which the target is derived. Primers and methods for multiplex amplification of dnaJ and a second target are also described.
Claims26
An oligonucleotide consisting of a target binding sequence selected from the group consisting of the target binding sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 and, optionally, a sequence required for an amplification reaction.
An oligonucleotide consisting of SEQ ID NO:8 or SEQ ID NO:9.
A method for amplifying a target nucleic acid sequence of the Mycobacterium avium complex comprising:
- a) hybridizing to the target nucleic acid
- i) a first amplification primer consisting of a target binding sequence selected from the group consisting of the target binding sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and, optionally, a sequence required for an amplification reaction, and
- ii) a second amplification primer consisting of a target binding sequence selected from the group consisting of the target binding sequences of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 and, optionally, the sequence required for the amplification reaction, and;
- b) extending the hybridized first and second amplification primers on the target nucleic acid sequence whereby the target nucleic acid sequence is amplified.
A method for amplifying a target nucleic acid sequence of the Mycobacterium avium complex comprising:
- a) hybridizing to the target nucleic acid
- i) a first amplification primer consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and
- ii) a second amplification primer consisting of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, and;
- b) amplifying the target nucleic acid in a Strand Displacement Amplification reaction.
An oligonucleotide selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:1, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
A method for simultaneously amplifying a first and a second target comprising:
- a) hybridizing a first amplification primer to the first target, the first amplification primer comprising the target binding sequence of SEQ ID NO:1 or SEQ ID NO:6 and a recognition site for a restriction endonuclease capable of nicking one strand of a double-stranded hemimodified recognition site for the restriction endonuclease, extending the first amplification primer to produce a first extension product and displacing the first extension product;
- b) hybridizing to the first extension product a first adapter primer consisting of the target binding sequence of SEQ ID NO:6 or the target binding sequence of SEQ ID NO:1 and a first adapter sequence substantially identical to a target binding sequence of a second amplification primer which hybridizes to the second target, the second amplification primer further comprising the recognition site for the restriction endonuclease, extending the first adapter primer to produce a second extension product and displacing the second extension product;
- c) hybridizing the second amplification primer to the second target, extending the second amplification primer to produce a third extension product and displacing the third extension;
- d) hybridizing to the third extension product a second adapter primer consisting of a target binding sequence which hybridizes to the third extension product and a second adapter sequence consisting of the target binding sequence of SEQ ID NO:1 or the target binding sequence of SEQ ID NO:6, extending the second adapter primer to produce a fourth extension product, displacing the fourth extension product and;
- e) simultaneously amplifying the second and fourth extension products using the first and second amplification primers.
A method for simultaneously amplifying a first and a second target comprising:
- a) hybridizing to the first target a first adapter primer consisting of the target binding sequence of SEQ ID NO:6 or the target binding sequence of SEQ ID NO:1 and a first adapter sequence substantially identical to a target binding sequence of a first amplification primer which hybridizes to the second target, the first amplification primer further comprising the recognition site for the restriction endonuclease, extending the first adapter primer to produce a first extension product and displacing the first extension product;
- b) hybridizing a second amplification primer to the first extension product, the second amplification primer comprising the target binding sequence of SEQ ID NO:1 or SEQ ID NO:6 and a recognition site for a restriction endonuclease capable of nicking one strand of a double-stranded hemimodified recognition site for the restriction endonuclease, extending the first amplification primer to produce a second extension product and displacing the second extension product;
- c) hybridizing to the second target a second adapter primer consisting of a target binding sequence which hybridizes to the second target and a second adapter sequence consisting of the target binding sequence of SEQ ID NO:1 or the target binding sequence of SEQ ID NO:6, extending the second adapter primer to produce a third extension product and displacing the third extension product;
- d) hybridizing the first amplification primer to the third extension product, extending the first amplification primer to produce a fourth extension product, displacing the fourth extension product and;
- e) simultaneously amplifying the second and fourth extension products using the first and second amplification primers.
Dependencies are inferred from the claim text.