A novel in situ polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues.
An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample (n=20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3'-3-diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.
Cardoso
Tereza C
TC
São Paulo State University, Laboratório de Virologia e Patologia Animal, Curso de Medicina Veterinária, Campus de Araçatuba, São Paulo, Brazil. tcardoso@fmva.unesp.br
Gomes
Deriane E
DE
Ferrari
Heitor F
HF
Silva-Frade
Camila
C
Rosa
Ana C G
AC
Andrade
Alexandre L
AL
Luvizotto
Maria Cecília R
MC
D000818:Animals; D001921:Brain / Q000821:virology; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D018792:Encephalitis, Viral / Q000662:veterinary* / Q000821:virology; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006566:Herpesviridae Infections / Q000662:veterinary* / Q000821:virology; D025881:Herpesvirus 5, Bovine / Q000235:genetics / Q000302:isolation & purification*; D017403:In Situ Hybridization / Q000379:methods*; D008590:Meningoencephalitis / Q000662:veterinary* / Q000821:virology; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
D017931:DNA Primers; D005404:Fixatives; D005557:Formaldehyde