Beggs M L, Cave M D, Marlowe C, Cloney L, Duck P, Eisenach K D
Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, USA.
J Clin Microbiol. 1996 Dec;34(12):2985-9. doi: 10.1128/jcm.34.12.2985-2989.1996.
Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110.
循环探针技术（CPT）是一种独特且简单的检测特定靶序列的方法。CPT利用一种嵌合的DNA-RNA-DNA探针，当与互补靶序列结合时可提供一个对核糖核酸酶H敏感的可切割连接。在本研究中，开发了一种基于CPT的诊断检测方法，用于检测结核分枝杆菌中的36bp直接重复序列（DR）区域。为了确定通过CPT利用DR检测结核分枝杆菌的可行性，使用三种DR探针通过Southern印迹杂交对多种分枝杆菌进行检测，以验证其特异性。对牛分枝杆菌401的整个DR区域进行了测序，并将数据用于设计一种PCR检测方法，使我们能够估计多种菌株中存在的DR数量。开发了一种使用与DR区域互补的探针的CPT检测方法，并用分枝杆菌的合成靶标和基因组DNA进行了评估。总之，36bp的DR为检测结核分枝杆菌提供了一个有吸引力的靶标，因为该序列在基因组中以高拷贝数存在，对结核分枝杆菌复合群具有特异性，并且在缺乏IS6110的菌株中也能发现。
