Open AccessQuantitative Fluorogenic PCR Assay for Measuring Ovine Herpesvirus 2 Replication in Sheep
Author(s) -
Daniela Hüssy,
Norbert Stäuber,
Christian M. Leutenegger,
Stefan Rieder,
Mathias Ackermann
Publication year - 2001
Publication title -
clinical and diagnostic laboratory immunology
Language(s) - English
Resource type - Journals
eISSN - 1098-6588
pISSN - 1071-412X
DOI - 10.1128/cdli.8.1.123-128.2001
Subject(s) - dna , biology , genomic dna , real time polymerase chain reaction , microbiology and biotechnology , polymerase chain reaction , primer dimer , virology , virus , dna replication , gene , genetics , multiplex polymerase chain reaction
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.