Validation of a dual-probe quantitative reverse transcription polymerase chain reaction for rapid detection of spring viremia of carp virus

Accurate and sensitive disease diagnosis is crucial to prevent the global spread of spring viremia of carp virus (SVCV). In this study, we developed and validated a novel dual-probe quantitative reverse transcription polymerase chain reaction (RT-qPCR) method, which detects two distinct SVCV genomic sequences. The analytical sensitivity of the developed method was 6.2 copies of the SVCV plasmid (LoD_95%). No cross-reactivity was observed with other viruses and fish cell lines. The diagnostic performance was further assessed using 550 koi carp (Cyprinus carpio) with a confirmed SVCV infection status. Compared to the conventional RT-PCR method, the dual-probe RT-PCR method diagnostic sensitivity (Dsn) and specificity (Dsp) of 100 % and 100 %, respectively. Additionally, when tested with 266 koi carp samples with an unconfirmed SVCV infection status, the novel method demonstrated a Dsn of 100 % and Dsp of 54.5 %, when compared to the of conventional RT-PCR method. No significant differences in inter- and intra-laboratory reproducibility were observed when performing PCR-based diagnosis using the novel method. Overall, the dual-probe RT-qPCR method targeting SVCV was found to be reliable tool for detecting the virus, suggesting its potential use in quarantine, surveillance, and monitoring programs.