{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T17:11:13Z","timestamp":1788369073230,"version":"build-2803163510"},"reference-count":14,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,11,29]],"date-time":"2025-11-29T00:00:00Z","timestamp":1764374400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["NRF-2023R1A2C1008182"],"award-info":[{"award-number":["NRF-2023R1A2C1008182"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008787","name":"National Institute of Fisheries Science","doi-asserted-by":"publisher","award":["R2025054"],"award-info":[{"award-number":["R2025054"]}],"id":[{"id":"10.13039\/501100008787","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Spring viremia of carp (SVC) is a highly contagious disease that affects cyprinids, resulting in systemic hemorrhage, abdominal distension, exophthalmia, and high mortality in juveniles. This can lead to significant losses in the aquaculture industry. The World Organization for Animal Health (WOAH) recommends a two-step semi-nested reverse transcription polymerase chain reaction (RT-PCR) method for diagnosis. However, this method is labor-intensive, requires large reagent volumes, and is prone to carry-over contamination. Here, we evaluated the detection sensitivity of one-step semi-nested RT-PCR (combining RT and primary amplification in a single tube, followed by a second nested PCR step) against conventional two-step semi-nested RT-PCR. SVC virus (SVCV) subgroup Ia was tested using cell culture, RT-quantitative PCR, and one-step RT-PCR. The two-step semi-nested PCR method detected viral RNA up to a 10\u22122 dilution, whereas one-step semi-nested RT-PCR detected it up to a 10\u22125 dilution, showing a 1000-fold improvement in sensitivity. Moreover, detection rates increased from 84.2% with two-step semi-nested RT-PCR to 91.7% with one-step semi-nested RT-PCR in fish tissue samples. One-step semi-nested RT-PCR reduces processing time, minimizes handling steps, and contamination risk, and enhances analytical sensitivity. This supports its adoption as a practical, high-throughput diagnostic tool for SVCV and consideration for future WOAH guidelines.<\/jats:p>","DOI":"10.3390\/microorganisms13122727","type":"journal-article","created":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T15:03:13Z","timestamp":1764687793000},"page":"2727","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Improved Detection Sensitivity of Spring Viremia of Carp Virus by Substituting a Two-Step with a One-Step Nested Reverse Transcription Polymerase Chain Reaction Method"],"prefix":"10.3390","volume":"13","author":[{"given":"Ji-Yoon","family":"Park","sequence":"first","affiliation":[{"name":"Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"In-Joo","family":"Shin","sequence":"additional","affiliation":[{"name":"Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyunwoo","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eun Sup","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Euna","family":"Choi","sequence":"additional","affiliation":[{"name":"WOAH Reference Laboratory for VHS, National Institute of Fisheries Science, Pusan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9123-4456","authenticated-orcid":false,"given":"Hyoung Jun","family":"Kim","sequence":"additional","affiliation":[{"name":"WOAH Reference Laboratory for VHS, National Institute of Fisheries Science, Pusan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4656-2143","authenticated-orcid":false,"given":"Se Ryun","family":"Kwon","sequence":"additional","affiliation":[{"name":"Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"261","DOI":"10.3354\/dao052261","article-title":"Spring viremia of carp (SVC)","volume":"52","author":"Ahne","year":"2002","journal-title":"Dis. Aquat. Organ."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1099\/jgv.0.000436","article-title":"Spring viraemia of carp virus: Recent advances","volume":"97","author":"Ashraf","year":"2016","journal-title":"J. Gen. Virol."},{"key":"ref_3","unstructured":"World Organisation for Animal Health (WOAH) (2023). Infection with spring viraemia of carp virus. Manual of Diagnostic Tests for Aquatic Animals, WOAH. Available online: https:\/\/www.woah.org\/fileadmin\/Home\/fr\/Health_standards\/aahm\/current\/2.3.09_SVC.pdf."},{"key":"ref_4","first-page":"514","article-title":"Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus","volume":"53","author":"Sanders","year":"2003","journal-title":"Comp. 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Methods."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"203","DOI":"10.3354\/dao053203","article-title":"Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups","volume":"53","author":"Stone","year":"2003","journal-title":"Dis. Aquat. Organ."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"263","DOI":"10.7853\/kjvs.2014.37.4.263","article-title":"One step multiplex RT-PCR preventing DNA carryover contamination for differential diagnosis of swine influenza viruses","volume":"37","author":"Kim","year":"2014","journal-title":"Korean J. Vet. 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Fish Dis."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Park, J.-Y., Kim, J.S., Kim, H.J., Choi, E., Kim, D.-H., Yun, D., Kasai, H., Nagata, J., Jung, T.S., and Kwon, S.R. (2026). Validation of a dual-probe quantitative reverse transcription polymerase chain reaction for rapid detection of spring viremia of carp virus. Aquaculture, in press.","DOI":"10.1016\/j.aquaculture.2025.743131"},{"key":"ref_13","unstructured":"World Trade Organization (WTO) (1995). Agreement on the Application of Sanitary and Phytosanitary Measures (SPS Agreement), WTO. Available online: https:\/\/www.wto.org\/english\/tratop_e\/sps_e\/spsagr_e.htm."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Zhang, N.Z., Zhang, L.F., Jiang, Y.N., Zhang, T., and Xia, C. (2009). Molecular analysis of spring vireamia of carp virus in China: A fatal aquatic viral disease that might spread in East Asia. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0006337"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/12\/2727\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T15:41:57Z","timestamp":1764690117000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/12\/2727"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,29]]},"references-count":14,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["microorganisms13122727"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms13122727","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,29]]}}}