D-1130. Simultaneous Sequence Analysis of Two Loci to Identify Mycobacterium spp.
Session: Poster Session: Laboratory Testing for Mycobacteria
Sunday, October 26, 2008: 12:00 AM
Room: Hall C
Background: The 16S rRNA gene is commonly used to identify Mycobacterium spp. in the clinical laboratory, but sequencing multiple DNA targets can provide better species resolution. Sequence Decoder [SD][iSentio, Bergen, Norway] is a software program that decodes mixed electropherograms enabling a laboratory to perform multi-locus sequencing in a single tube. We evaluated the ability of SD to simultaneously analyze 16S rRNA and rpoB gene sequences for mycobacterial identification. Methods:
Isolates identified as Mycobacterium spp. by partial 16S rRNA sequencing at ARUP Laboratories were obtained. Amplification was performed on lysates with primers targeting 500bp of 16S rRNA and 700bp of rpoB genes. Sequencing was performed with forward and reverse primers for each target. Electropherograms were analyzed with SD. Final identification was determined by separate phylogenetic analyses of individual16S rRNA and rpoB sequences. Results: Of 55 Mycobacterium spp., 52 were decoded by SD, and identified as M. abscessus (13), M. gordonae (7), M. avium (4), M. mucogenicum (4), M. fortuitum (3), M. lentiflavum (3), M. peregrinum (3), M. immunogenum (2), M. intracellulare/chimaera (2), M. kansasii (2), M. chelonae (2), M. avium complex variant (1), M. chelonae variant (1), M. interjectum (1), M. massiliense (1), M. mageritense (1), M. phocaicum (1), and M. tuberculosis complex (1). Three sequences failed to produce sufficient signal for analysis. For 30 isolates, low interspecies variability existed with the 16S rRNA gene, and rpoB sequence data was necessary for species resolution. For 4 isolates, 16S rRNA data was required for identification due to lack of rpoB reference sequences in Genbank. Conclusions: Sequence Decoder was an effective tool for simultaneous analysis of dual loci sequences of 16S rRNA and rpoB genes. This application provided greater resolution to species, eliminating the need for reflex testing to discriminate between closely related species.
Bjarte Karlsen1, Cathy Petti, MD2, Keith Simmon, Masters3, ystein Saebo1, yvind Kommedal, MD1 and  K. E. Simmon,
iSentio Role(s): Consultant, Received: Consulting Fee., (1)iSentio, (2)University of Utah School of Medicine, Salt Lake City, UT, (3)ARUP, Salt Lake City, UT