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.

Fig. 1

Phylogenetic tree based on the nucleotide sequences of the capsid protein genes of 15 FCV isolates used in the study and the F-9 strain (GenBank accession number M86379)

Fig. 2

Standard curve obtained with tenfold serial dilutions from 10⁻⁴ to 10⁻⁹ of standard RNA (generated from the threshold cycle values) plotted against the logarithmic concentration of the serial dilutions