Development and application of a triplex TaqMan RT-PCR assay for simultaneous detection of Feline calicivirus, Feline Herpesvirus 1 and Feline parvovirus

Abstract The feline calicivirus (FCV), feline herspesvirus 1 (FHV-1) and feline panleukemia virus (FPV) are heavily threaten the health of cats. In this study, a triplex TaqMan real-time polymerase chain reaction (RT-PCR) assay (triplex assay) was developed to detect these viruses. The optimized concentration of primers was 0.5 µM of each, probes concentration was 0.1 µM for FCV and FHV-1, 0.05 µM for FPV. The annealing temperature was set at 54 ℃. The triplex RT-PCR assay was carefully validated. The detection limit for FPV, FCV, and FHV-1 was 5×101 copies/µL, which showed a 10-100-fold increase in the sensitivity compared with the conventional PCR. The coefficients of variation (CV) of the intra-assay variability of the test were < 1.86%, and that of inter-assay was < 3.19%, indicating excellent repeatability and reproducibility of the triplex assay. Additionally, the assay has perfect specificity. In a pilot study, samples from 48 cats were analyzed using the triplex RT-PCR method and the commercial kits, and further confirmed by sequencing. The positive rates for the samples analyzed with these two methods were 70.83% and 62.5%, respectively, which demonstrated that the developed method was more accurate than the commercial kits in clinical diagnosis.