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Nucleic Acids Research 2005 33(15):4978-4986; doi:10.1093/nar/gki814
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Published online 2 September 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Adam P. Silverman and Eric T. Kool*

Department of Chemistry, Stanford University Stanford, CA 94305-5080, USA

*To whom correspondence should be addressed. Tel: +1 650 724 4741; Fax: +1 650 725 0259; Email: eric.kool{at}stanford.edu

Received June 6, 2005. Revised August 17, 2005. Accepted August 17, 2005.

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.

Dedicated to Professor Peter Dervan on his 60th birthday.


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