NUCLEOTIDE-SEQUENCE AND STRAND DISPLACEMENT AMPLIFICATION OF THE 70K PROTEIN GENE FROM MYCOBACTERIA

We isolated and determined the nucleotide sequence of the 70K gene fro m nine mycobacteria and from two related non-mycobacteria with the goa l of obtaining a region of requisite specificity to serve as a mycobac terial genus-specific probe. Two different primer sets were then desig ned to amplify the 70K gene using strand displacement amplification. U sing one of the primer sets, 10 different mycobacteria were readily de tected with sensitivities of 100 molecules DNA, and with only cross-re activity to two non-mycobacteria. The other set of primers that were t ested amplified the same set of mycobacteria, but exhibited no crossre activity with non-mycobacterial DNAs. By employing one of the primer s ets, we were able to successfully amplify with high sensitivity three different target DNA sequences comprised of the 70K mycobacterial genu s target, an IS 6110 (M. tuberculosis complex) target, and an internal amplification control using SDA. These results demonstrate the potent ial of the 70K gene to serve as a mycobacterial genus-specific probe, and demonstrate the first multiplex amplification by SDA of three DNA targets.