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Applied and Environmental Microbiology, June 2003, p. 3317-3326, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3317-3326.2003

Fluorescent Heteroduplex Assay for Monitoring Bacillus anthracis and Close Relatives in Environmental Samples

Lori Merrill, Jennifer Richardson, Cheryl R. Kuske, and John Dunbar^*

Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico

Received 30 September 2002/ Accepted 5 March 2003

A fluorescent heteroduplex method was developed to assess the presence of 16S rRNA gene (rDNA) sequences from Bacillus anthracis and close relatives in PCR-amplified 16S rDNA sequence mixtures from environmental samples. The method uses a single-stranded, fluorescent DNA probe, 464 nucleotides in length, derived from a B. anthracis 16S rRNA gene. The probe contains a unique, engineered deletion such that all probe-target duplexes are heteroduplexes with an unpaired G at position 343 (G343). Heteroduplex profiles of sequences 85% similar to the probe were produced using an ABI 377 sequencer in less than 3 h. The method divides strains of the Bacillus cereus-Bacillus thuringiensis-B. anthracis group into two subgroups. Each subgroup is defined by a specific 16S rRNA gene sequence type. Sequence type A, containing one mismatch with the probe, occurs in B. anthracis and a small number of closely related clonal lineages represented mostly by food-borne pathogenic isolates of B. cereus and B. thuringiensis. Sequence type B, containing two mismatches with the probe, is found in the majority of B. cereus and B. thuringiensis strains examined to date. Sequence types A and B, when hybridized to the probe, generate two easily differentiated heteroduplexes. Thus, from heteroduplex profiles, the presence of B. cereus-B. thuringiensis-B. anthracis subgroups in environmental samples can be inferred unambiguously. The results show that fluorescent heteroduplex analysis is an effective profiling technique for detection and differentiation of sequences representing small phylogenetic or functional groups in environmental samples.

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* Corresponding author. Mailing address: M888 Biosciences Div., Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 667-8806. Fax: (505) 665-3024. E-mail: dunbar{at}lanl.gov.

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Applied and Environmental Microbiology, June 2003, p. 3317-3326, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3317-3326.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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