Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.
Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.
Walker
G T
GT
Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016.
Nadeau
J G
JG
Spears
P A
PA
Schram
J L
JL
Nycz
C M
CM
Shank
D D
DD
D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D012045:Regulatory Sequences, Nucleic Acid
D004269:DNA, Bacterial