Development of an allele-specific quantitative polymerase chain reaction assay for differentiating the RLB 106 strain from the wild-type viruses of Varicellovirus bovinealpha1

Varicellovirus bovinealpha1 (BoAHV1) is an important causal agent of various pathological conditions, such as respiratory and reproductive diseases, in cattle. An intranasal vaccine containing a temperature-sensitive mutant RLB 106 strain is used to control BoAHV1-related diseases. To monitor the invasion of BoAHV1 wild-type viruses in vaccinated populations, it is essential to develop a simple method for differentiating between the RLB 106 strain and BoAHV1 wild-type viruses. In this study, we developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) assay that targets the point mutation G23136A located in UL40 for detecting the RLB 106 strain. We calculated the difference in Cq values (ΔCq) obtained from a paired qPCR using each point mutation site-targeting primer set and established the cutoff ΔCq for differentiation. The accuracy of differentiation was confirmed by the DNA partial sequencing of UL40. Results demonstrated that differentiation by AS-qPCR was consistent with the results obtained by DNA sequencing, and the field isolates classified as the RLB 106 strain exhibited a temperature-sensitive phenotype. Hence, the AS-qPCR assay developed in this study could be a promising method for the simple differentiation between the RLB 106 strain and wild-type viruses in a single-step reaction.