Development of a TaqMan Probe‐Based Quantitative PCR Assay Targeting the N Gene for Detection of Carpione rhabdovirus 2023

ABSTRACT The Carpione rhabdovirus strain 2023 (CAPRV2023) is an emerging viral pathogen that has caused significant morbidity and mortality in cultured golden pompano ( Trachinotus ovatus ) in China over recent years. There is an urgent need for a rapid and accurate method for the quantitative detection of CAPRV2023. In this study, we developed a TaqMan probe‐based quantitative PCR assay targeting the N gene of CAPRV2023 and systematically evaluated its specificity, sensitivity, repeatability, and reproducibility of the assay. Both plasmid DNA, in vitro‐transcribed RNA standards, and in vitro‐transcribed RNA mixed with RNA extracted from CAPRV2023‐negative golden pompano tissues were used as templates to evaluate assay performance under both optimal and biologically relevant conditions. Using plasmid DNA standards, the assay exhibited excellent linearity over a broad range of 2 × 10 1 to 5 × 10 9 copies/μL, with a standard curve of Y = −3.3132 X + 39.142, a correlation coefficient of R 2 = 0.9993, an amplification efficiency of 100.37%, and a detection limit of 20 copies per reaction. In vitro‐transcribed RNA standards demonstrated robust linearity over the range of 2 × 10 2 to 2 × 10 9 copies/μL, with a standard curve of Y = −3.2327 X + 45.502, a correlation coefficient of R 2 = 0.9968, and a detection limit of 200 copies per reaction. Notably, when the in vitro‐transcribed RNA standards were combined with RNA extracted from CAPRV2023‐negative golden pompano tissues, the assay maintained similar performance, yielding a standard curve of Y = −3.2182 X + 45.445, a correlation coefficient of R 2 = 0.9967, and the same detection limit of 200 copies per reaction. These results indicate that the presence of background tissue RNA does not significantly interfere with assay accuracy or sensitivity. Specificity test revealed that the assay exhibits no cross‐reactivity with common bacterial or viral agents present in various aquatic organisms. The assay demonstrated high reproducibility and repeatability, with intra‐assay and inter‐assay coefficients of variation (CVs) below 2.5%. Field sample detection yielded a significantly higher detection rate compared to the conventional PCR assay. The newly developed TaqMan qPCR assay provides a robust diagnostic tool for the efficient and accurate quantitative detection of CAPRV2023 in field samples and surveillance programs.