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Clinical Chemistry 50: 702-708, 2004. First published February 12, 2004; 10.1373/clinchem.2003.026781
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Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2004;50:702-708.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay

Marcus Panning1, Marcel Asper1, Stefanie Kramme1, Herbert Schmitz1 and Christian Drosten1,a

1 Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

aAddress correspondence to this author at: Bernhard Nocht Institute for Tropical Medicine, Department of Virology, Bernhard-Nocht Strasse 74, 20359 Hamburg, Germany. Fax 49-40-42818-378; e-mail drosten{at}bni-hamburg.de.

Background: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes.

Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction.

Results: Strain differentiation results showed little interassay variability (CV, 0.4–0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with >=95% probability (235–849 genome copies/mL of plasma).

Conclusions: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.




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