Detection of mycobacteria by multiplex nucleic acid amplification

A method for simultaneously amplifying multiple Mycobacterium target sequences and an internal control sequence in a sample comprising:

• a) adding an internal control sequence consisting of SEQ ID NO:
• 12 to the sample;
• b) hybridizing a first primer consisting of SEQ ID NO:
• 1 to the target sequences, extending the first primer with polymerase to produce a first extension product and displacing the first extension product;
• b) hybridizing a second primer consisting of SEQ ID NO:
• 2 to the first extension product, extending the second primer to produce a second extension product and displacing the second extension product;
• c) hybridizing a third primer consisting of SEQ ID NO:
• 5 to the target sequences, extending the third primer with polymerase to produce a third extension product and displacing the third extension product;
• d) hybridizing a fourth primer consisting of SEQ ID NO:
• 6 to the third extension product, extending the fourth primer with polymerase to produce a fourth extension product and displacing the fourth extension product, and;
• e) simultaneously amplifying the second and fourth extension products and the internal control sequence in a Strand Displacement Amplification reaction using SEQ ID NO:
• 1 and SEQ ID NO:
• 5 as amplification primers.

An oligonucleotide consisting of a sequence selected from the group consisting of SEQ ID NO:

• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 5 and SEQ ID NO:
• 6.

An oligonucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:

• 13, SEQ ID NO:
• 14, SEQ ID NO:
• 15, SEQ ID NO:
• 16, SEQ ID NO:
• 17, SEQ ID NO:
• 18 and SEQ ID NO:
• 19.

A method for genus-specific amplification of Mycobacterium target sequences in a sample comprising:

• a) hybridizing a first primer consisting of SEQ ID NO:
• 5 to the target sequences, extending the first primer with polymerase to produce a first extension product and displacing the first extension product;
• b) hybridizing a second primer consisting of SEQ ID NO:
• 6 to the first extension product, extending the second primer to produce a second extension product and displacing the second extension product, and;
• e) amplifying the second extension product in a Strand Displacement Amplification reaction using SEQ ID NO:
• 1 and SEQ ID NO:
• 5 as amplification primers.

A kit for amplifying Mycobacterium target sequences comprising:

• a) a first oligonucleotide consisting of SEQ ID NO:
• 1, a second oligonucleotide consisting of SEQ ID NO:
• 2, a third oligonucleotide consisting of SEQ ID NO:
• 5 and a fourth oligonucleotide consisting of SEQ ID NO:
• 6, and;
• b) reagents for hybridizing the first, second, third and fourth oligonucleotides to the Mycobacterium target sequences, extending the oligonucleotides and displacing the oligonucleotides from the target sequences in an amplification reaction.

A kit for genus-specific amplification of Mycobacterium target sequences comprising:

• a) a first oligonuclectide consisting of SEQ ID NO:
• 5 and a second oligonucleotide consisting of SEQ ID NO:
• 6, and
• b) reagents for hybridizing the first and second oligonucleotides to the Mycobacterium target sequences, extending the oligonucleotides and displacing the oligonucleotides from the target sequences in an amplification reaction.