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Journal of Clinical Microbiology, June 2002, p. 1985-1988, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.1985-1988.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection of Smallpox Virus DNA by LightCycler PCR

Mark J. Espy,1 Franklin R. Cockerill III,1,2 Richard F. Meyer,3 Michael D. Bowen,3 Gregory A. Poland,4 Ted L. Hadfield,5 and Thomas F. Smith1*

Division of Clinical Microbiology,1 Division of Infectious Diseases,2 Division of General Internal Medicine, Mayo ClinicFoundation; Rochester, Minnesota,4 Centers for Disease Control and Prevention, Atlanta, Georgia,3 Armed Forces Institute of Pathology, Walter Reed Army Medical Center, Washington, D.C.5

Received 19 March 2002/ Accepted 20 March 2002

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [Tm], 56.40°C), monkeypox virus (Tm, 56.24°C), and vaccinia virus (Tm, 56.72°C), including the Dryvax vaccine strain, from smallpox virus (Tm, 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.

* Corresponding author. Mailing address: Division of Clinical Microbiology, Mayo Clinic, 200 First St., SW, Rochester, MN 55905. Phone: (507) 284-2255. Fax: (507) 284-4272. E-mail: tfsmith{at}mayo.edu.

Journal of Clinical Microbiology, June 2002, p. 1985-1988, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.1985-1988.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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