Clinical Chemistry
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Clinical Chemistry 52: 316-319, 2006; 10.1373/clinchem.2005.060335
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(Clinical Chemistry. 2006;52:316-319.)
© 2006 American Association for Clinical Chemistry, Inc.

Technical Briefs

Real-Time PCR Detection of Parapoxvirus DNA,

Andreas Nitsche1,a, Mathias Büttner2, Sonja Wilhelm3, Georg Pauli1 and Hermann Meyer4

1 Robert Koch-Institut, Zentrum für Biologische Sicherheit 1, Berlin, Germany;2 Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Oberschleissheim, Germany;3 Institut für Tierhygiene und Öffentliches Veterinärwesen, Leipzig, Germany;4 Bundeswehr Institute of Microbiology, München, Germany;

aaddress correspondence to this author at: Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany; fax 49-30-4547-2605, e-mail nitschea{at}rki.de


Abstract

Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported.

Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms.

Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed.

Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.




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