Effects of sampling instrument and processing technique on DNA yield and detection rate for feline herpesvirus-1 via polymerase chain reaction assay

Abstract

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. Sample Population - Crandell-Rees feline kidney (CRFK) cells grown in vitro and conjunctival samples from 40 eyes of 20 cats. Procedures - Samples of CRFK cells (collected by use of a swab or cytology brush, with or without suspension in FBS 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 sus pended 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-PCH assay. Conclusions and Clinical Relevance - The DV-PCR assay of DNA extracted from an unsuspended swab sample was th preferred method for assessment of conjunctival shedding of FHV-1 in cats.