This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Olson, V. A. Articles by Meyer, H. Search for Related Content PubMed PubMed Citation Articles by Olson, V. A. Articles by Meyer, H.

Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus

Poxvirus Section, Centers for Disease Control and Prevention, Atlanta, Georgia,¹ Artus Biotech,² Bernhard Nocht Institute for Tropical Medicine, Hamburg,⁴ Robert Koch Institut, Berlin,⁵ Institute of Microbiology of the German Armed Forces, Munich, Germany,⁶ State Research Center of Virology and Biotechnology ("Vector"), Koltsovo, Russia³

Received 15 September 2003/ Returned for modification 31 October 2003/ Accepted 21 January 2004

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus Orthopoxvirus. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (T_ms) determined for 46 strains of variola virus (T_ms, 55.9 to 57.8°C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (T_ms, 61.7 to 62.7°C), 15 strains of monkeypox virus (T_ms, 61.9 to 62.2°C), 40 strains of cowpox virus (T_ms, 61.3 to 63.7°C), 8 strains of mousepox virus (T_m, 61.9°C), and 8 strains of camelpox virus (T_ms, 64.0 to 65.0°C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.

This article has been cited by other articles:

• Kurth, A., Achenbach, J., Miller, L., Mackay, I. M., Pauli, G., Nitsche, A. (2008). Orthopoxvirus Detection in Environmental Specimens during Suspected Bioterror Attacks: Inhibitory Influences of Common Household Products. Appl. Environ. Microbiol. 74: 32-37 [Abstract] [Full Text]  
• Vanpouille, C., Biancotto, A., Lisco, A., Brichacek, B. (2007). Interactions between Human Immunodeficiency Virus Type 1 and Vaccinia Virus in Human Lymphoid Tissue Ex Vivo. J. Virol. 81: 12458-12464 [Abstract] [Full Text]  
• Scaramozzino, N., Ferrier-Rembert, A., Favier, A.-l., Rothlisberger, C., Richard, S., Crance, J.-M., Meyer, H., Garin, D. (2007). Real-Time PCR to Identify Variola Virus or Other Human Pathogenic Orthopox Viruses. Clin. Chem. 53: 606-613 [Abstract] [Full Text]  
• Sulaiman, I. M., Tang, K., Osborne, J., Sammons, S., Wohlhueter, R. M. (2007). GeneChip Resequencing of the Smallpox Virus Genome Can Identify Novel Strains: a Biodefense Application. J. Clin. Microbiol. 45: 358-363 [Abstract] [Full Text]  
• Fedele, C. G., Negredo, A., Molero, F., Sanchez-Seco, M. P., Tenorio, A. (2006). Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans. J. Clin. Microbiol. 44: 4464-4470 [Abstract] [Full Text]  
• Niedrig, M., Meyer, H., Panning, M., Drosten, C. (2006). 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. 44: 1283-1287 [Abstract] [Full Text]  
• Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
• Karem, K. L., Reynolds, M., Braden, Z., Lou, G., Bernard, N., Patton, J., Damon, I. K. (2005). Characterization of Acute-Phase Humoral Immunity to Monkeypox: Use of Immunoglobulin M Enzyme-Linked Immunosorbent Assay for Detection of Monkeypox Infection during the 2003 North American Outbreak. CVI 12: 867-872 [Abstract] [Full Text]