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Journal of Clinical Microbiology, December 2006, p. 4464-4470, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.00276-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans{triangledown}

C. G. Fedele,1,2* A. Negredo,2 F. Molero,2 M. P. Sánchez-Seco,1,2 and A. Tenorio2

Alerts and Emergencies Unit, National Center of Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain,1 Arbovirus and Imported Viral Diseases Laboratory, Diagnostic Microbiology Service, National Center of Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain2

Received 8 February 2006/ Returned for modification 10 April 2006/ Accepted 6 October 2006

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.

* Corresponding author. Mailing address: National Center of Microbiology, Instituto de Salud Carlos III, Ctra. Pozuelo Majadahonda Km 2, 28220 Majadahonda, Madrid, Spain. Phone: 34-91-8223405. Fax: 34-91-5097919. E-mail: gfedele{at}isciii.es.

{triangledown} Published ahead of print on 25 October 2005.

Journal of Clinical Microbiology, December 2006, p. 4464-4470, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.00276-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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