Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR.
A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R(2) = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.
Vögtlin
A
A
Institute of Virology, University of Zurich, Zurich, Switzerland.
Fraefel
C
C
Albini
S
S
Leutenegger
C M
CM
Schraner
E
E
Spiess
B
B
Lutz
H
H
Ackermann
M
M
D000818:Animals; D001082:Aqueous Humor / Q000821:virology*; D002371:Cat Diseases / Q000821:virology*; D002415:Cats; D003236:Conjunctivitis, Viral / Q000662:veterinary / Q000821:virology; D004279:DNA, Viral / Q000032:analysis*; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000662:veterinary / Q000821:virology; D016849:Keratitis, Herpetic / Q000662:veterinary / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism
D004279:DNA, Viral; D019914:Taq Polymerase