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Analysis of Ba813 DNA sequence from several Bacillus sp isolates

Analysis of Ba813 DNA sequence from several Bacillus sp isolates

Abstracts of the General Meeting of the American Society for Microbiology 103: H-045

Real-time polymerase chain reaction (RT-PCR) of the genetic marker Ba813 is widely considered a reliable indicator for the identification of B. anthracis isolates, along with other genes located on the pX01 and pX02 plasmids. Detailed ecological and evolutionary relationships of closely related species such as Bacillus cereus and Bacillus thuringiensis to Bacillus anthracis are largely unknown and difficult to determine. This is compounded by the fact that several B. cereus have been shown to contain the Ba813 chromosomal marker. It has been known in recent years that several B. cereus isolates contain the Ba813 chromosomal marker. Sequencing this chromosomal marker from a wide variety of "non-anthracis" isolates will make possible the elucidation of Ba813 representation in closely related Bacillus isolates. Using the Primer Select application of the Lasergene (DNAStar, Inc., Madison, WI) software package, primers specific to the Ba813 chromosomal marker were developed. PCR and sequencing were carried out using a Biometra (Horsham, PA) 96-well T-gradient thermocycler. Raw sequence data was obtained using a Beckman-Coulter CEQ 2000XL (Fullerton, CA) DNA sequencer. Finally, sequence data was assembled and BLAST searched using the Seqman application of Lasergene. Four B. cereus Ba813 sequences had sequence identity of 96.4%. (267 of 277 base pairs) to 98.4% (244 of 248 base pairs) compared to the Ba813 sequence of B. anthracis. The primers used to obtain the data above produced multiple banding patterns for 9 of the Bacillus isolates. Subsequent tests showed that several of these "multi-band" Bacillus isolates tested positive by RT-PCR for Ba813 by both ABI 7700 (Applied Biosystems, Inc., Foster City, CA) and LightCycler (Roche, Indianapolis, IN) using in-house designed primers and those of Center for Disease Control and Prevention (CDC) even though gel and sequencing analysis to date have yielded no Ba813 sequence data. In conclusion, at least four of the B. cereus isolates contain the Ba813 genomic marker, which may interfere with typing strategies, based on the presence or absence of Ba813.

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