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

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

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Bibliographic Details
Published in:	American journal of veterinary research Vol. 69; no. 6; pp. 811 - 817
Main Authors:	Westermeyer, H.D, Kado-Fong, H, Maggs, D.J
Format:	Journal Article
Language:	English
Published:	United States 01-06-2008
Subjects:	accuracy Animals cat diseases Cat Diseases - diagnosis Cat Diseases - virology Cats Cell Line cell lines Conjunctival Diseases - diagnosis Conjunctival Diseases - veterinary Conjunctival Diseases - virology disease detection disease diagnosis disease severity DNA DNA, Viral - chemistry DNA, Viral - genetics DNA, Viral - isolation & purification extraction Felid herpesvirus 1 hepatocytes Herpesviridae - genetics Herpesviridae - isolation & purification Herpesviridae Infections - diagnosis Herpesviridae Infections - veterinary Herpesviridae Infections - virology in vitro studies keratinocytes polymerase chain reaction Polymerase Chain Reaction - veterinary processing equipment sampling Specimen Handling - instrumentation Specimen Handling - veterinary validity viral diseases of animals and humans
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Summary:	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. Crandell-Rees feline kidney (CRFK) cells grown in vitro and conjunctival samples from 40 eyes of 20 cats. 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. 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. 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0002-9645 1943-5681
DOI:	10.2460/ajvr.69.6.811