Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus

Abstract

Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats and is associated with interstitial pneumonia, oral ulceration and polyarthritis. Recently, outbreaks have involved a highly virulent FCV that leads to multisystemic signs. Virus isolation and conventional RT-PCR are the most common methods used for FCV diagnosis. However, real-time RT-PCR offers a rapid, sensitive, specific and easy tool for nucleic acid detection. The objective of this study was to design a TaqMan probe-based, real-time RT-PCR assay for detection of FCV. It was determined in our previous study that the first 120 nucleotides of the 5' region of the genome are highly conserved among FCV isolates. Primers and a probe specific for this region were designed for a real-time RT-PCR assay to detect FCV. Initial validation was done using 15 genetically diverse isolates. Also, 122 samples were tested by the new assay and virus isolation. The real-time RT-PCR assay was as sensitive and specific as virus isolation and was far more rapid. This real-time RT-PCR assay targeting the conserved 5' region of the genome is a fast, economical and accurate method for detection of FCV.

Correlation between clinical signs of feline conjunctivitis and molecular detection of felid herpesvirus-1, feline calicivirus, chlamydophila felis and mycoplasma felis in cats from shelters in Rio de Janeiro

“…Therefore, it is possible to infer that, although not apparently capable of determining clinical conjunctivitis alone, these bacteria may play an important role complicating pre-existing infections as has been suggested by other authors (SYKES et al, 2001;HARTMANN et al, 2010). Our data suggest that, for clinical diagnosis of conjunctivitis by FHV-1, FCV, C.felis, and M.felis infections, symptoms overlap and therefore diagnosis has to be confirmed by molecular testing A high genetic variability is expected for FCV (ABD-ELDAIM et al, 2009;BERGER et al, 2015) and the specificity of the PCR primers may have been a limitation of the study.…”

Feline calicivirus and other respiratory pathogens in cats with Feline calicivirus-related symptoms and in clinically healthy cats in Switzerland

“…For FCV, two previously described FCV real-time TaqMan RT-qPCR assays were used [ 25 , 26 ]. The assays were optimized prior to the start of the experiment.…”

“…The temperature profile was 30 min at 48 °C, followed by 10 min at 95 °C and 45 cycles of 15 s at 95 °C, followed by 1 min at 60 °C. The FCV RT-qPCR S2 [ 26 ] reaction contained 0.5 μL Superscript III/RT Platinum Taq Mix (Invitrogen, Life technologies), 300 nM forward primer, 900 nM reverse primer, 250 nM probe, 0.05 μL 2.5x Rox dye, 0.625 μL 40 U/μL rRNasin® RNase Inhibitor (Promega, Dübendorf, Switzerland) or RNasin® Plus RNase Inhibitor (Promega) and 3.95 μL nuclease-free water (Gibco, Life Technologies). The temperature profile was 30 min at 50 °C, followed by 2 min at 95 °C and 45 cycles of 20 s at 95 °C, followed by 45 s at 60 °C.…”

Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

“…Feline caliciviruses (FCV), a member of the genus Vesivirus in the family Caliciviridae, is an important pathogen and cause upper respiratory tract diseases in cats. Prevalence of FCV was 29.4% in clinically diseased cats (Abd-Eldaim et al, 2009), and 43.1% in cat population of a rescue shelter (Zicola et al, 2009). At present, vaccination is administered worldwide to prevent FCV infection in pet cats.…”