DETECTION OF MYCOBACTERIUM-TUBERCULOSIS DNA WITH THERMOPHILIC STRAND DISPLACEMENT AMPLIFICATION AND FLUORESCENCE POLARIZATION

Strand displacement amplification (SDA) is an isothermal, in vitro met hod for diagnostics that amplifies a target DNA sequence by using a re striction enzyme and DNA polymerase. We have combined a new thermophil ic form of SDA that involves restriction enzyme BsoBI and polymerase e xo(-) Bca with fluorescence polarization for detection of Mycobacteriu m tuberculosis DNA by using the IS6110 insertion element as the target sequence, A 5'-fluorescein-labeled oligodeoxynucleotide detector prob e hybridizes to the amplified product as it rises in concentration dur ing SDA, and the single- to double-stranded conversion is monitored th rough an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected wi th an extremely simple protocol that takes only 20 min and uses relati vely simple instrumentation and reagents, all of which can be purchase d off-the-shelf.