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Clinical and Diagnostic Laboratory Immunology, January 2001, p. 123-128, Vol. 8, No. 1
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.1.123-128.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Quantitative Fluorogenic PCR Assay for Measuring Ovine Herpesvirus 2 Replication in Sheep

D. Hüssy,1 N. Stäuber,1 C. M. Leutenegger,2 S. Rieder,3 and M. Ackermann1,*

Institute of Virology1 and Department of Internal Veterinary Medicine,2 University of Zurich, and Institute of Animal Science, Swiss Federal Institute of Technology,3 Zurich, Switzerland

Received 13 July 2000/Returned for modification 25 September 2000/Accepted 11 October 2000

A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.

* Corresponding author. Mailing address: Institute of Virology, Veterinary Medical Faculty, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland. Phone: 41 1 635 87 01. Fax: 41 1 635 89 11. E-mail: ma{at}vetvir.unizh.ch.

Clinical and Diagnostic Laboratory Immunology, January 2001, p. 123-128, Vol. 8, No. 1
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.1.123-128.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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