Comparison of three restriction endonucleases in IS 1245-based RFLP typing of Mycobacterium avium

Abstract

IS1245-based restriction fragment length polymorphism (RFLP) analysis has beenproposed recently for molecular typing of Mycobacteriurn 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, sekcted 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.

Comparison of antimycobacterial activity of grepafloxacin against Mycobacterium avium with that of levofloxacin: accumulation of grepafloxacin in human macrophages

“…The isolated strains were identified as M. avium, using DNA-DNA hybridization (DDH) of mycobacterium (Kyokuto, Tokyo, Japan) and by detecting the insertion sequence, IS1245, using the polymerase chain reaction. 12 M. avium type strain, ATCC 25291, was obtained from the American Type Culture Collection (Manassas, VA, USA).…”

Related Strains ofMycobacterium aviumCause Disease in Children with AIDS and in Children with Lymphadenitis

“…Strains were grown and DNA isolated as above. DNA was digested with BsaA1 [17], run on an 0.8% agarose gel, transferred by Southern blotting to a nylon membrane, and probed with a biotin-labeled PCR-generated fragment that encoded a portion of IS1245 (nucleotides 135-561) [9]. Targets were detected by use of the enhanced chemiluminescence labeling and detection systems (Amersham, Buckinghamshire, United Kingdom).…”

Unusual high-performance liquid chromatography profile of a strain of Mycobacterium avium

“…The IS 1245 ‐based restriction fragment length polymorphism analysis [2,3] of both isolates yielded different patterns (Figure 2), thus demonstrating the double origin of the infection.…”