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Nucleic Acids Research 2005 33(2):e13; doi:10.1093/nar/gni015
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Published online 19 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 2 © Oxford University Press 2005; 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 permissions, please contact journals.permissions{at}oupjournals.org.

Methods Online

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Douglas Horejsh*, Federico Martini, Fabrizio Poccia, Giuseppe Ippolito, Antonino Di Caro and Maria R. Capobianchi

National Institute for Infectious Diseases ‘Lazzaro Spallanzani’–IRCCS Rome, Italy

*To whom correspondence should be addressed at INMI L. Spallanzani, Virologia-Baglivi/Del Vecchio, via Portuense 292, 00149 Rome, Italy. Tel: +39 06 551 70907; Fax: +39 06 551 70918; Email: horejsh{at}inmi.it

Received September 2, 2004. Revised October 18, 2004. Accepted December 22, 2004.

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.


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