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Applied and Environmental Microbiology, March 2009, p. 1614-1620, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02120-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Assay for Detection of a New Simulant for Poxvirus Biothreat Agents{triangledown}

Laurence Garnier,* Jean-Christophe Gaudin, Paul Bensadoun,{dagger} Isabelle Rebillat, and Yannick Morel

Délégation Générale pour l'Armement, Centre d'Études du Bouchet, 5 Rue Lavoisier, 91710 Vert le Petit, France

Received 12 September 2008/ Accepted 5 January 2009

Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant "simulant" microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the Cydia pomonella granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per µl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows Cydia pomonella granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies.

* Corresponding author. Mailing address: Délégation Générale pour l'Armement, Centre d'Expertise Parisien, Sciences de l'Homme et Protection, 7-9 Rue des Mathurins, 92220 Bagneux, France. Phone: (33) 1 46 19 76 05. Fax: (33) 1 46 19 75 90. E-mail: laurence.garnier{at}dga.defense.gouv.fr

{triangledown} Published ahead of print on 23 January 2009.

{dagger} Present address: IRBM-INSERM U841, Laboratoire de Virologie Moléculaire et Immunologie-Physiopathologie et Thérapeutique des Hépatites Virales Chroniques, 51 Avenue du Mal de Lattre de Tassigny, 94010 Créteil, France.

Applied and Environmental Microbiology, March 2009, p. 1614-1620, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02120-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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