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Quantitative real time polymerase chain reaction (q RT‐PCR ) and RNA scope in situ hybridization ( RNA ‐ ISH ) as effective tools to diagnose feline herpesvirus‐1‐associated dermatitis
Author(s) -
Mazzei Maurizio,
Vascellari Marta,
Zanardello Claudia,
Melchiotti Erica,
Vannini Susanna,
Forzan Mario,
Marchetti Veronica,
Albanese Francesco,
Abramo Francesca
Publication year - 2019
Publication title -
veterinary dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.744
H-Index - 60
eISSN - 1365-3164
pISSN - 0959-4493
DOI - 10.1111/vde.12787
Subject(s) - real time polymerase chain reaction , in situ hybridization , microbiology and biotechnology , messenger rna , rna , polymerase chain reaction , chemistry , biology , biochemistry , gene
Background Felid herpesvirus type 1 ( FHV ‐1)‐associated dermatitis is characterized by facial and nasal involvement; clinical and histopathological manifestations may overlap with other dermatitides. Objective To evaluate the realibility of q RT‐PCR ‐2 − ΔΔC q and RNA scope in situ hybridization ( RNA ‐ ISH ) methods to diagnose FHV ‐1‐associated dermatitis, in formalin‐fixed paraffin‐embedded ( FFPE ) tissues. Animals Sixteen FFPE samples from cats with facial dermatitis and four controls were studied. Methods and materials Based on histopathological features, cases were separated into: Group 1, samples with herpetic dermatitis (four); Group 2, samples with nonherpetic facial dermatitis (six); Group 3, samples with facial dermatitis of ambiguous nature (allergic or viral) (six); and Group 4, samples from healthy cats (four). A relative quantification using the 2 − ΔΔC q method was used to estimate the “upregulation” of each FHV ‐1 target viral gene copies (glycoprotein‐B and thymidine‐kinase) relative to reference gene. Detection of FHV ‐1 mRNA was performed using the RNA scope 2.5 detection kit. Results By 2 − ΔΔC q analysis, upregulation of both FHV ‐1 genes was observed in all samples from Group 1 and two of six from Group 3. No upregulation was identified in samples from groups 2 and 4. Positive mRNA hybridization signal was observed in all cases from Group 1 and two cases of Group 3. No positivity was observed in samples from groups 2 and 4. Conclusions and clinical importance Q RT‐PCR 2 −ΔΔCq analysis and RNA ‐ ISH can identify the FHV ‐1 genome as causative agent of the associated dermatitis, even where inclusion bodies are not detectable. Both techniques are functional in retrospective studies, have greater specificity than conventional PCR , and may be proposed for research and diagnostic purposes.