Nucleotide sequence and strand displacement amplification of the 70K protein gene from mycobacteria

We isolated and determined the nucleotide sequence of the 70K gene from nine mycobacteria and from two related non-mycobacteria with the goal of obtaining a region of requisite specificity to serve as a mycobacterial genus-specific probe. Two different primer sets were then designed to amplify the 70K gene using strand displacement amplification. Using one of the primer sets, 10 different mycobacteria were readily detected with sensitivities of 100 molecules DNA, and with only cross-reactivity to two non-mycobacteria. The other set of primers that were tested amplified the same set of mycobacteria, but exhibited no crossreactivity with non-mycobacterial DNAs. By employing one of the primer sets, we were able to successfully amplify with high sensitivity three different target DNA sequences comprised of the 70K mycobacterial genus target, an IS 6110 (M. tuberculosis complex) target, and an internal amplification control using SDA. These results demonstrate the potential of the 70K gene to serve as a mycobacterial genus-specific probe, and demonstrate the first multiplex amplification by SDA of three DNA targets.