Little M C, Spears P A, Shank D D
Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016.
Mol Cell Probes. 1994 Oct;8(5):375-84. doi: 10.1006/mcpr.1994.1054.
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.
我们从9种分枝杆菌和2种相关的非分枝杆菌中分离并测定了70K基因的核苷酸序列，目的是获得具有必要特异性的区域，用作分枝杆菌属特异性探针。然后设计了两套不同的引物，使用链置换扩增法扩增70K基因。使用其中一套引物，能够轻松检测出10种不同的分枝杆菌，灵敏度可达100个DNA分子，且仅与两种非分枝杆菌有交叉反应。测试的另一套引物扩增出了相同的分枝杆菌，但与非分枝杆菌DNA无交叉反应。通过使用其中一套引物，我们能够成功地以高灵敏度扩增出由70K分枝杆菌属靶标、IS 6110（结核分枝杆菌复合群）靶标和使用链置换扩增法的内部扩增对照组成的三种不同的靶DNA序列。这些结果证明了70K基因作为分枝杆菌属特异性探针的潜力，并证明了首次通过链置换扩增法对三个DNA靶标进行多重扩增。
