{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T20:03:53Z","timestamp":1779912233840,"version":"3.53.1"},"reference-count":52,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T00:00:00Z","timestamp":1767744000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Scientific Research Platforms and Projects of Regular Institutions of Higher Education in Guangdong Province","award":["2025ZDZX4019"],"award-info":[{"award-number":["2025ZDZX4019"]}]},{"name":"Innovation Program in Postgraduate Education of Guangdong Ocean University","award":["202526"],"award-info":[{"award-number":["202526"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Carpione rhabdovirus (CAPRV) is an emerging virus within the family Rhabdoviridae, posing potential threats to aquaculture species such as golden pompano (Trachinotus anak). However, since the 21st century, and for CAPRV strains isolated from marine fish, only a single CAPRV2023 sequence has previously been available in public databases, with no additional sequences reported. Because the virus undergoes genetic variation, relying on this single sequence likely introduced mismatches or off-target risks in earlier detection assay designs. Notably, the previously developed two-step N-targeting detection assay was designed based solely on that single CAPRV2023 sequence. Consequently, this study involved determining and analyzing the N gene sequences from CAPRV isolates gathered from 2023 to 2025, with the aim of pinpointing conserved regions for assay development, and sequence comparisons subsequently verified the existence of mismatches in the primer\u2013probe binding sites of the previous assay. Since quantitative assays in aquatic virology often define copy numbers utilizing either plasmid DNA templates or RNA templates produced via in vitro transcription, which may lead to variations in amplification kinetics and sensitivity, this study compared both standards to ensure reliable quantification across different nucleic acid types. Based on these findings, a one-step TaqMan quantitative PCR (qPCR) assay was developed and validated using dual nucleic acid standards, namely plasmid DNA and in vitro\u2013transcribed RNA. Compared with conventional two-step qPCR, the one-step format combines cDNA synthesis and subsequent DNA amplification in a single sealed tube, thereby effectively preventing cross-contamination, simplifying the workflow, and improving detection efficiency. The assay exhibited strong linearity (R2 &gt; 0.99) and consistent amplification efficiencies between 90% and 110%, demonstrating excellent quantitative performance. The detection limits were 2 copies per reaction for plasmid DNA and 20 copies for in vitro\u2013transcribed RNA templates. No cross-reactivity was observed with other aquatic pathogens, and the assay showed strong repeatability and reproducibility (coefficients of variation below 2.0%), providing a sensitive and reliable tool for epidemiological surveillance and the analysis of CAPRV distribution in marine aquaculture systems of southern China.<\/jats:p>","DOI":"10.3390\/microorganisms14010126","type":"journal-article","created":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T09:34:12Z","timestamp":1767778452000},"page":"126","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Overcoming Target Drift: Development and Validation of a One-Step TaqMan qPCR Assay for Epidemiological Surveillance of Carpione rhabdovirus Circulating in Southern China"],"prefix":"10.3390","volume":"14","author":[{"given":"Yucong","family":"Huang","sequence":"first","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6811-8876","authenticated-orcid":false,"given":"Zhiyuan","family":"Huang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoyu","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojuan","family":"Li","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Liu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huajian","family":"Lin","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Animal Disease Prevention and Control Center, Guangzhou 510230, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Animal Disease Prevention and Control Center, Guangzhou 510230, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaofeng","family":"Chen","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jichang","family":"Jian","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6443-1369","authenticated-orcid":false,"given":"Heng","family":"Sun","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture & Key Laboratory of Control for Diseases of Aquatic Economic Animals\u00a0of Guangdong Higher Education Institutes, Fisheries College\u00a0of Guangdong Ocean University, Zhanjiang 524088, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,7]]},"reference":[{"key":"ref_1","unstructured":"PRC (2025). 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