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Received on October 1, 2003
Accepted on January 29, 2004
Molecular Diagnostics and Genetics |
1 Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
* To whom correspondence should be addressed. 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.
The following articles in journals at HighWire Press have cited this article:
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  S. Kramme, L. Van An, N. D. Khoa, L. Van Trin, E. Tannich, J. Rybniker, B. Fleischer, C. Drosten, and M. Panning Orientia tsutsugamushi Bacteremia and Cytokine Levels in Vietnamese Scrub Typhus Patients J. Clin. Microbiol., March 1, 2009; 47(3): 586 - 589. [Abstract] [Full Text] [PDF]
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 A. Kurth, J. Achenbach, L. Miller, I. M. Mackay, G. Pauli, and A. Nitsche Orthopoxvirus Detection in Environmental Specimens during Suspected Bioterror Attacks: Inhibitory Influences of Common Household Products Appl. Envir. Microbiol., January 1, 2008; 74(1): 32 - 37. [Abstract] [Full Text] [PDF]
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C. G. Fedele, A. Negredo, F. Molero, M. P. Sanchez-Seco, and A. Tenorio Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans J. Clin. Microbiol., December 1, 2006; 44(12): 4464 - 4470. [Abstract] [Full Text] [PDF]
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 M. R. Savona and P. J. Danaher Vaccinia DNA in Blood After Smallpox Vaccination--Reply JAMA, September 20, 2006; 296(11): 1351 - 1352. [Full Text] [PDF]
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M. R. Savona, W. P. Dela Cruz, M. S. Jones, J. A. Thornton, D. Xia, T. L. Hadfield, and P. J. Danaher Detection of vaccinia DNA in the blood following smallpox vaccination. JAMA, April 26, 2006; 295(16): 1898 - 1900. [Full Text] [PDF]
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M. Niedrig, H. Meyer, M. Panning, and C. Drosten Follow-Up on Diagnostic Proficiency of Laboratories Equipped To Perform Orthopoxvirus Detection and Quantification by PCR: the Second International External Quality Assurance Study J. Clin. Microbiol., April 1, 2006; 44(4): 1283 - 1287. [Abstract] [Full Text] [PDF]
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J. Dreier, M. Stormer, and K. Kleesiek Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays J. Clin. Microbiol., September 1, 2005; 43(9): 4551 - 4557. [Abstract] [Full Text] [PDF]
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D. P. Fedorko, J. C. Preuss, G. A. Fahle, L. Li, S. H. Fischer, P. Hohman, and J. I. Cohen Comparison of Methods for Detection of Vaccinia Virus in Patient Specimens J. Clin. Microbiol., September 1, 2005; 43(9): 4602 - 4606. [Abstract] [Full Text] [PDF]
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