This web site is provided as a free service to advance the identification of Mycobacterium species through the use of partial sequencing of the 65 Kilodalton Heat Shock Protein gene using a database constructed from two published manuscripts (J. Clin. Microbiol. 2004; 42:3000-3011. and J. Clin. Microbiol. 2006; 44:60-66). The primary deterrents of using hsp65 partial gene sequencing as a method for identifying mycobacteria reside with the need for a comprehensive database and determining the differential percentage match between clinical isolates and valid species or groups. A recent analysis of organisms isolated by our laboratory indicated a 99.1% correlation between hsp65 based identifications and our standard algorithm of commercial probes, traditional biochemical test panels and reference identification by partial 16S rRNA gene sequencing. This indicates that our current database is sufficient to correctly identify organisms from our geographical local, but the user must be aware that it may not be as discriminatory with isolates from other geographical locations. In our experience the first distinct hsp65 sequence match exceeding 97% with our database entries usually represents a correct identification. Exceptions exist however and we prefer a match exceeding 98% with a species or taxon to be considered a correct identification. A match of less than 97% indicates that the isolate does not belong to any entry in the database and should be reported as a unique Mycobacterium species. An equal match with two species or taxons usually indicates that the sequence does not belong to either species, represents a new taxon or species and should be reported as a unique Mycobacterium species. There are several entries in the database that are characteristic of species by biochemical test panels, but can not be assigned to that species by hsp65 sequence. These are represented in the database as species variants (for example M. terrae var VM 372) and should be reported as the biochemically defined species (example M. terrae) until further taxonomic clarification occurs.

The current version of the database also contains limited sequence entries for some Nocardia and Tsukamurella species. Nocardia hsp65 sequence entries are based on a limited number of type strains sequenced in our laboratory as well as clinical isolates identified by partial 16S rRNA gene sequences following the criterion of Mellmann et al. (Int. J. Med. Microbiol. 2003; 293: 359-370.) and then partially sequencing the hsp65 gene. We have not set cutoff limit criteria for Nocardia species, but believe that an identical match with a type strain sequence is differential. Any percentage match less than 100% should be reported as Nocardia species. Tsukamurella sequences are based on type strains sequenced by our laboratory and our analysis indicates that only a match of 100% with a type strain sequence can be considered to be a valid identification. Any match with a Tsukamurella species lower than 100% should be reported only as Tsukamurella species. Please be aware that evolutionary drift of the hsp65 and the 16S rRNA genes may not allow direct identification comparison of isolates. If information regarding valid Mycobacterium species is required please link out to http://www.bacterio.cict.fr/. If interpretation of BLAST report results is required please email Alan McNabb, Molecular Services, BC Centre for Disease Control.

Click here to begin hsp65 BLAST Search. New to BLAST? Link out to the NCBI FAQ on using BLAST.

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