Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis
Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis
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DOI：
10.1128/jcm.39.4.1595-1599.2001
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发表时间：
2001-04-01
影响因子：
9.4
通讯作者：
Rastogi, N
中科院分区：
文献类型：
--
作者：
Legrand, E;Filliol, I;Rastogi, N
基于II型结核分枝杆菌复合菌DR基因座43个间隔区的变异性，间隔区寡核苷酸分型是一种快速的方法，有助于结核病流行病学的研究。由于在间隔区31和32之间插入额外拷贝的IS 6110，这可能导致引物靶的不对称分裂，因此导致属于间隔区寡聚型50的结核病临床分离株（I. Filliol，C，Sola和N. Rastogi，J，Clin，Microbiol. 38：1231-1234，2000）。在本研究中，将先前的观察结果扩展到50型的25个临床分离株，表明选择性扩增左侧和中心DR区的引物组IS 6-DRb确实能够证明间隔区31的存在。IS 6110-限制性片段长度多态性（RFLP）和DR-RFLP分析表明，50型分离株的特征是存在两个与DR位点相关的IS 6110拷贝和一个额外的1.4kb的IS 6110双带。然后使用引物组IS 3-IS 6选择性地扩增DR位点内750 bp的IS 6110片段。中心DR区的测序证实了我们先前的发现，并表明在50型分离株中间隔区31的缺失是由于间隔区31和32之间额外拷贝的IS 6110的不对称插入，导致DRa-DRb靶不均等地分裂成两个部分，分别为6和30 bp。这些结果表明，DR基因座构成理想的IS 6110优先基因座（ipl），允许插入两个或更多个。为IS 6110-RFLP和Spoligotyping谱的变化提供了流行病学和系统发育学解释的新线索。
Based on the variability of 43 spacers within the direct repeat (DR) locus of Il Mycobacterium tuberculosis complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis, It was recently hypothesized that despite its presence in the DR locus, spacer 31 could not be amplified in M. tuberculosis clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS6110 between spacers 31 and 32 that could lead to an asymmetrical split of the primer targets (I. Filliol, C, Sola, and N. Rastogi, J, Clin, Microbiol. 38:1231-1234, 2000), In the present investigation, previous observations were extended to 25 clinical isolates of type 50 showing that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of spacer 31. IS6110-restriction fragment length polymorphism (RFLP and DR-RFLP showed that type 50 isolates were characterized by the presence of two copies of IS6110 associated with the DR locus and an additional double IS6110 band of 1,4 kb, The primer set IS3-IS6 was then used to selectively amplify a 750-bp inter-IS6110 fragment within the DR locus. The sequencing of the central DR region corroborated our previous findings and showed that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS6110 between spacers 31 and 32, leading to an unequal split of the DRa-DRb target into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS6110 preferential locus (ipl), permitting the insertion of two or. more copies of IS6110, and provide new clues for epidemiological and phylogenetic interpretation of changes in IS6110-RFLP and spoligotyping profiles.