{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T19:29:20Z","timestamp":1793734160060,"version":"4.6.1"},"reference-count":20,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,6,29]],"date-time":"2021-06-29T00:00:00Z","timestamp":1624924800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,6,29]],"date-time":"2021-06-29T00:00:00Z","timestamp":1624924800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Virol J"],"published-print":{"date-parts":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                <jats:title>Background<\/jats:title>\n                <jats:p>Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (<jats:italic>EF1\u03b1<\/jats:italic>). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: <jats:italic>R<\/jats:italic><jats:sup>2<\/jats:sup>\u2009=\u20090.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0\u2009\u2265\u2009threshold cycle value, and the virus-specific peak higher than the <jats:italic>EF1\u03b1<\/jats:italic>-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12985-021-01591-3","type":"journal-article","created":{"date-parts":[[2021,6,29]],"date-time":"2021-06-29T12:02:42Z","timestamp":1624968162000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y"],"prefix":"10.1186","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0674-7538","authenticated-orcid":false,"given":"Nobuya","family":"Onozuka","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takehiro","family":"Ohki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norikuni","family":"Oka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tetsuo","family":"Maoka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,6,29]]},"reference":[{"key":"1591_CR1","unstructured":"FAOSTAT. The database on production of crops. 2018 (online database). http:\/\/www.fao.org\/faostat\/en\/#data\/QC."},{"key":"1591_CR2","volume-title":"Compendium of potato diseases","year":"2001","unstructured":"Stevenson WR, Loria R, Franc GD, Weingartner DP, editors. Compendium of potato diseases. 2nd ed. St Paul: APS Press; 2001.","edition":"2"},{"key":"1591_CR3","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-030-28683-5","volume-title":"The potato crop its agricultural, nutritional and social contribution to humankind","author":"H Campos","year":"2020","unstructured":"Campos H, Ortiz O. The potato crop its agricultural, nutritional and social contribution to humankind. New York: Springer; 2020. https:\/\/doi.org\/10.1007\/978-3-030-28683-5."},{"key":"1591_CR4","unstructured":"USDA. Canada\/US-Management Plan for potato viruses that cause tuber necrosis. 2004 (Online document). https:\/\/www.aphis.usda.gov\/plant_health\/plant_pest_info\/potato\/downloads\/pvy\/NecroticVirusManagementPlan.pdf."},{"key":"1591_CR5","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1270\/jsbbs.65.17","volume":"65","author":"T Kawakami","year":"2015","unstructured":"Kawakami T, Oohori H, Tajima K. Seed potato production system in Japan, staring from foundation seed of potato. Breed Sci. 2015;65:17\u201325.","journal-title":"Breed Sci"},{"key":"1591_CR6","unstructured":"UNECE. UNECE standard S-1 seed potatoes, 2018 edn. 2018. (Online document). https:\/\/www.unece.org\/fileadmin\/DAM\/trade\/agr\/standard\/potatoes\/S-1_SeedPotatoes_2018_E.pdf."},{"key":"1591_CR7","unstructured":"AuSPICA. Conditions for the AuSPICA seed potato certification scheme. 2019. (Online document). https:\/\/www.auspica.org.au\/wp-content\/uploads\/2020\/02\/AuSPICA-Scheme-condition-2019.pdf."},{"key":"1591_CR8","unstructured":"SASA. Scottish seed potato classification scheme. Explanatory leaflet. 2020. https:\/\/www.sasa.gov.uk\/document-library\/growing-crop-leaflet."},{"key":"1591_CR9","first-page":"37","volume":"99","author":"R Kumar","year":"2017","unstructured":"Kumar R, Jeevalatha A, Baswaraj R, Sharma S, Nagesh M. A multiplex RT-PCR assay for simultaneous detection of five viruses in potato. J Plant Pathol. 2017;99:37\u201345.","journal-title":"J Plant Pathol"},{"key":"1591_CR10","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1016\/S0166-0934(00)00140-3","volume":"86","author":"X Nie","year":"2000","unstructured":"Nie X, Singh RP. Detection of multiple potato viruses using an Oligo(dT) as a common cDNA primer in multiplex RT-PCR. J Virol Methods. 2000;86:179\u201385.","journal-title":"J Virol Methods"},{"key":"1591_CR11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.jviromet.2006.12.012","volume":"142","author":"BO Agindotan","year":"2007","unstructured":"Agindotan BO, Shiel PJ, Berger PH. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR. J Virol Methods. 2007;142:1\u20139.","journal-title":"J Virol Methods"},{"key":"1591_CR12","doi-asserted-by":"publisher","first-page":"379","DOI":"10.1007\/s00705-012-1493-6","volume":"158","author":"J Cheng","year":"2013","unstructured":"Cheng J, Jiang Y, Rao P, Wu H, Dong Q, Wu Z, Ding X, Guo J. Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen. Arch Virol. 2013;158:379\u201386.","journal-title":"Arch Virol"},{"key":"1591_CR13","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1007\/s10327-020-00923-5","volume":"86","author":"N Onozuka","year":"2020","unstructured":"Onozuka N, Ohki T, Oka N, Maoka T. Detection of four major potato viruses in Japan using a simple RNA preparation and one-step multiplex RT-PCR. J Gen Plant Pathol. 2020;86:290\u20139.","journal-title":"J Gen Plant Pathol"},{"key":"1591_CR14","doi-asserted-by":"publisher","first-page":"51","DOI":"10.15446\/agron.colomb.v34n1.53161","volume":"34","author":"CD Vallejo","year":"2016","unstructured":"Vallejo CD, Guti\u00e9rrez SPA, Mar\u00edn MM. Genome characterization of a potato virus S (PVS) variant from tuber sprouts of Solanum phureja Juz. et Buk. Agron Colomb. 2016;34:51\u201360.","journal-title":"Agron Colomb"},{"key":"1591_CR15","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1016\/j.virol.2017.03.018","volume":"507","author":"KJ Green","year":"2017","unstructured":"Green KJ, Brown CJ, Gray SM, Karasev AV. Phylogenetic study of recombinant strains of potato virus Y. Virology. 2017;507:40\u201352.","journal-title":"Virology"},{"key":"1591_CR16","doi-asserted-by":"publisher","first-page":"15431","DOI":"10.1073\/pnas.1001454107","volume":"107","author":"JM Huguet","year":"2010","unstructured":"Huguet JM, Bizarro CV, Forns N, Smith SB, Bustamante C, Ritort F. Single-molecule derivation of salt dependent base-pair free energies in DNA. PNAS. 2010;107:15431\u20136.","journal-title":"PNAS"},{"key":"1591_CR17","doi-asserted-by":"publisher","first-page":"42","DOI":"10.1016\/j.jviromet.2018.04.008","volume":"257","author":"M Aloisio","year":"2018","unstructured":"Aloisio M, Morelli M, Elicio V, Saldarelli P, Ura B, Bortot B, Severini GM, Minafra A. Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis. J Virol Methods. 2018;257:42\u20137.","journal-title":"J Virol Methods"},{"key":"1591_CR18","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1016\/j.jviromet.2004.10.005","volume":"123","author":"A Varga","year":"2005","unstructured":"Varga A, James D. Detection and differentiation of plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing. J Virol Methods. 2005;123:213\u201320.","journal-title":"J Virol Methods"},{"key":"1591_CR19","doi-asserted-by":"publisher","first-page":"380","DOI":"10.4149\/av_2015_04_380","volume":"59","author":"P Huo","year":"2015","unstructured":"Huo P, Shen WT, Yan P, Tuo DC, Li XY, Zhou P. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and mosaic virus by multiplex real-time reverse transcription PCR. Acta Virol. 2015;59:380\u20138.","journal-title":"Acta Virol"},{"key":"1591_CR20","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1111\/j.1365-2338.2005.00805.x","volume":"35","author":"A Fox","year":"2005","unstructured":"Fox A, Evans F, Browning I. Direct tuber testing for Potato Y Potyvirus by real-time RT-PCR and ELISA: reliable options for post-harvest testing? EPPO Bull. 2005;35:93\u20137.","journal-title":"EPPO Bull"}],"container-title":["Virology Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12985-021-01591-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12985-021-01591-3\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12985-021-01591-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,6,29]],"date-time":"2021-06-29T12:10:30Z","timestamp":1624968630000},"score":1,"resource":{"primary":{"URL":"https:\/\/virologyj.biomedcentral.com\/articles\/10.1186\/s12985-021-01591-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,29]]},"references-count":20,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2021,12]]}},"alternative-id":["1591"],"URL":"https:\/\/doi.org\/10.1186\/s12985-021-01591-3","relation":{},"ISSN":["1743-422X"],"issn-type":[{"value":"1743-422X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,29]]},"assertion":[{"value":"8 February 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 May 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"29 June 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Not applicable.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"131"}}