Fisanotti J C, Romano M I, Alito A, Bigi F, Cataldi A
Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria, Moron, Argentina.
Res Microbiol. 1997 Jun;148(5):427-35. doi: 10.1016/S0923-2508(97)83873-9.
A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.
在用感染牛的血清筛选牛分枝杆菌基因组文库的过程中，检测到一个携带含有mpb - 64基因和3'侧翼序列的质粒的克隆（质粒pMBA122）。当将pMBA122插入片段用作针对经PvuII消化的分枝杆菌DNA的Southern印迹杂交探针时，它能区分不同的结核分枝杆菌复合群物种。该探针与结核分枝杆菌中的一条7kb条带、牛分枝杆菌中的一条5kb条带以及野生海豹的结核分枝杆菌复合群菌株中的一条3kb条带杂交。Smal基因组消化使我们能够将这个多态性区域定位在mpb - 64基因的下游。为了克隆这个特定区域，我们设计了一对PCR引物。出乎意料的是，这些引物仅扩增出牛分枝杆菌DNA；在结核分枝杆菌DNA中未观察到扩增。当退火温度从70℃降至55℃时，用结核分枝杆菌获得了相同大小的扩增产物。该产物被克隆并测序，显示与牛分枝杆菌扩增片段有部分同源性。因此，该区域包含与结核分枝杆菌同源性低于其他比较序列的牛分枝杆菌序列。这表明使用针对该区域的PCR可以在物种水平上实现对结核分枝杆菌复合群更精确的区分方法。
