Published online before print April 10, 2008

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Copyright © 2008 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2008.070094

Accepted for publication December 18, 2007.

Article

Direct Sequence Detection of Structured H5 Influenza Viral RNA

Matthew B. Kerby^*, Sarah Freeman^*, Kristina Prachanronarong^*, Andrew W. Artenstein, Steven M. Opal, and Anubhav Tripathi^*@

From the Chemical and Biochemical Engineering Laboratory,* Biomedical Engineering Division of Engineering, Brown University; the Department of Medicine and Center for Biodefense and Emerging Pathogens, Memorial Hospital of Rhode Island; and the Warren Alpert Medical School, Brown University, Providence, Rhode Island

^@ To whom correspondence should be addressed. E-mail: anubhav_tripathi{at}brown.edu.

	Abstract

We describe the development of sequence-specific molecular beacons (dual-labeled DNA probes) for identification of the H5 influenza subtype, cleavage motif, and receptor specificity when hybridized directly with in vitro transcribed viral RNA (vRNA). The cloned hemagglutinin segment from a highly pathogenic H5N1 strain, A/Hanoi/30408/2005(H5N1), isolated from humans was used as template for in vitro transcription of sense-strand vRNA. The hybridization behavior of vRNA and a conserved subtype probe was characterized experimentally by varying conditions of time, temperature, and Mg²⁺ to optimize detection. Comparison of the hybridization rates of probe to DNA and RNA targets indicates that conformational switching of influenza RNA structure is a rate-limiting step and that the secondary structure of vRNA dominates the binding kinetics. The sensitivity and specificity of probe recognition of other H5 strains was calculated from sequence matches to the National Center for Biotechnology Information influenza database. The hybridization specificity of the subtype probes was experimentally verified with point mutations within the probe loop at five locations corresponding to the other human H5 strains. The abundance frequencies of the hemagglutinin cleavage motif and sialic acid recognition sequences were experimentally tested for H5 in all host viral species. Although the detection assay must be coupled with isothermal amplification on the chip, the new probes form the basis of a portable point-of-care diagnostic device for influenza subtyping.