Effects of sampling instrument and processing technique on DNA yield and detection rate for feline herpesvirus-1 via polymerase chain reaction assay.
OBJECTIVE
To assess effects of disease severity, sampling instrument, and processing technique on extracted DNA yield and detection rate for feline herpesvirus-1 (FHV-1) via PCR assay.


METHODS
Crandell-Rees feline kidney (CRFK) cells grown in vitro and conjunctival samples from 40 eyes of 20 cats.


METHODS
Samples of CRFK cells (collected by use of a swab or cytology brush, with or without suspension in PBS solution) underwent DNA extraction; DNA yield was quantified spectrophotometrically. In affected cats, signs of herpetic disease were subjectively assessed. Conjunctival swab and brush samples were collected bilaterally for measurement of DNA concentration; a defined mass (DM) of DNA and defined volume (DV) of sample were assessed for FHV-1 via PCR assays.


RESULTS
For CRFK cells, DNA yields from unsuspended swabs and brushes were greater than for suspended swabs and brushes; suspended swab samples yielded less DNA than suspended brush samples. For conjunctival samples, DNA yields from swabs were greater than for brushes. Clinical score was not correlated with double-stranded DNA yield collected via either sampling instrument; however, cats with FHV-1-positive assay results had higher clinical scores than cats with FHV-1-negative results. Detection of FHV-1 in swab and brush samples was similar. Double-stranded DNA yield and FHV-1 detection were inversely related via DM-PCR assay. The DV-PCR assay had a significantly higher FHV-1 detection rate than the DM-PCR assay.


CONCLUSIONS
The DV-PCR assay of DNA extracted from an unsuspended swab sample was the preferred method for assessment of conjunctival shedding of FHV-1 in cats.
Westermeyer
Hans D
HD
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Kado-Fong
Helen
H
Maggs
David J
DJ
D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis / Q000821:virology*; D002415:Cats; D002460:Cell Line; D003229:Conjunctival Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006564:Herpesviridae / Q000235:genetics* / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D013048:Specimen Handling / Q000295:instrumentation / Q000662:veterinary*
D004279:DNA, Viral