Amplification and detection of the alpha antigen gene of mycobacterium avium complex species
AssigneeBecton, Dickinson and Company
InventorsSilvia A. Bustos, Christine A. Rostkowski, Keith C. Williams, Leslie A. Stringfellow
Life of this patent
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Abstract
Oligonucleotide primers derived from the BCG-85B gene of mycobacteria and methods for complex-specific amplification of a fragment of the BCG-85B gene in species of the Mycobacterium avium Complex (MAC) are provided. Also disclosed are an oligonucleotide detector probe useful for detecting the amplification products of the BCG-85B gene in MAC species and adaptation of the primers for multiplex amplification reactions.
Claims25
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, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and, optionally, a sequence required for an amplification reaction.
An oligonucleotide selected from the group consisting of SEQ ID NO :11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18.
A method for amplifying a target nucleic acid 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:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 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:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and, optionally the sequence required for the amplification reaction, and;
- b) extending the hybridized first and second amplification primers on the target nucleic acid whereby the target nucleic acid is amplified.
A method for amplifying a target nucleic acid 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:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, and
- ii) a second amplification primer consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, and;
- b) amplifying the target nucleic acid in a Strand Displacement Amplification reaction.
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:2 and a recognition site for a restriction endonuclease which is 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:2 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 product;
- 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 substantially identical to the target binding sequence of SEQ ID NO:1 or the target binding sequence of SEQ ID NO:2, 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 a first amplification primer to the first target, the first amplification primer comprising the target binding sequence of SEQ ID NO:4 or SEQ ID NO:8 and a recognition site for a restriction endonuclease which is 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:8 or the target binding sequence of SEQ ID NO:4 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 product;
- 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 substantially identical to the target binding sequence of SEQ ID NO:4 or the target binding sequence of SEQ ID NO:8, 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.
Dependencies are inferred from the claim text.