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 met hod of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multi plex form of SDA that allows two target sequences and an internal ampl ification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of ta rget fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. W e applied multiplex SDA to co-amplification of a target sequence (IS61 10) that is specific to members of the Mycobacterium tuberculosis-comp lex and a target (16S ribosomal gene) that is common to most clinicall y relevant species of mycobacteria. Both targets are amplified 10(8)-f old during a 2 hour, single temperature incubation. The relative sensi tivity of the system was evaluated across a number of clinically relev ant mycobacteria and checked for crossreactivity against organisms tha t are closely related to mycobacteria.