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Clinical Chemistry 53: 1899-1905, 2007. First published August 23, 2007; 10.1373/clinchem.2007.093245
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(Clinical Chemistry. 2007;53:1899-1905.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

Marit Kramski1,2, Helga Meisel1, Boris Klempa1,3, Detlev H. Krüger1, Georg Pauli2 and Andreas Nitsche2,a

1 Institute of Virology, Helmut-Ruska-Haus, Charité Campus Mitte, Berlin, Germany.
2 Robert Koch-Institut, Zentrum für Biologische Sicherheit 1, Berlin, Germany.
3 Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.

aAddress correspondence to this author at: Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany. Fax 49-(0)30-18754-2605; e-mail nitschea{at}rki.de.

Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens.

Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing.

Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice.

Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.




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J. A. Jordan, J. Jones-Laughner, and M. B. Durso
Utility of Pyrosequencing in Identifying Bacteria Directly from Positive Blood Culture Bottles
J. Clin. Microbiol., February 1, 2009; 47(2): 368 - 372.
[Abstract] [Full Text] [PDF]


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