Garzelli C, Lari N, Nguon B, Cavallini M, Pistello M, Falcone G
Department of Biomedicine, University of Pisa, Italy.
J Med Microbiol. 1997 Nov;46(11):933-9. doi: 10.1099/00222615-46-11-933.
IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.
最近有人提出基于IS1245的限制性片段长度多态性（RFLP）分析用于鸟分枝杆菌分离株的分子分型。由于在最佳限制酶方面没有标准化方法，因此测试了三种限制性内切酶对17株人分离株进行分析。这些限制性内切酶是根据IS1245和高度同源的IS1311的物理图谱选择的，分别是切割IS1245的BsaAI、切割IS1311的PvuII以及切割IS1245和IS1311的NruI。所有限制性内切酶都产生了多态性和复杂的RFLP图谱。然而，BsaAI和NruI产生的条带比PvuII产生的条带分布更均匀且更容易检测，PvuII产生的条带大多聚集在指纹图谱的一个狭窄区域。在某些情况下，用BsaAI或NruI进行DNA消化产生的探针特异性限制性片段的分子大小低于预期。此外，预期在所有分离株中产生最多条带的NruI消化产生的条带数量分别比BsaAI或PvuII在14株和5株分离株中产生的条带数量少。这些发现可能表明鸟分枝杆菌分离株中存在未鉴定的与IS1245相关的插入元件。对鸟分枝杆菌分离株基于IS1245的RFLP图谱进行计算机分析表明，尽管存在微小差异，但这些限制性内切酶能够定义不同的条带模式以及相同或高度相关分离株的聚类，从而证实基于IS1245的RFLP分析是一种用于流行病学研究的有用技术。
