{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,9]],"date-time":"2026-11-09T17:10:55Z","timestamp":1794244255401,"version":"4.9.0"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T00:00:00Z","timestamp":1759363200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0"},{"start":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T00:00:00Z","timestamp":1759363200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0"}],"funder":[{"DOI":"10.13039\/501100004748","name":"Mashhad University of Medical Sciences","doi-asserted-by":"publisher","award":["970708"],"award-info":[{"award-number":["970708"]}],"id":[{"id":"10.13039\/501100004748","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"DOI":"10.1038\/s41598-025-16972-9","type":"journal-article","created":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T09:34:09Z","timestamp":1759397649000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Optimization of the real-time PCR platform method using high-resolution melting analysis and comparison with sequencing and phylogenetic analysis for developing optimal malaria diagnostic methods"],"prefix":"10.1038","volume":"15","author":[{"given":"Omid","family":"Ahmadi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yousef","family":"Sharifi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Saeed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amirali","family":"Reihani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seyed Aliakbar","family":"Shamsian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elham","family":"Moghaddas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hadi","family":"Mirahmadi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soudabeh","family":"Etemadi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reza","family":"Fotouhi-Ardakani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mehdi","family":"Zarean","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,10,2]]},"reference":[{"key":"16972_CR1","doi-asserted-by":"publisher","first-page":"F1000-Faculty","DOI":"10.12688\/f1000research.14991.1","volume":"7","author":"M Recker","year":"2018","unstructured":"Recker, M., Bull, P. C. & Buckee, C. O. Recent advances in the molecular epidemiology of clinical malaria. F1000Research. 7, F1000-Faculty (2018).","journal-title":"F1000Research."},{"key":"16972_CR2","doi-asserted-by":"publisher","first-page":"3339","DOI":"10.2147\/IDR.S405668","volume":"16","author":"M Fikadu","year":"2023","unstructured":"Fikadu, M. & Ashenafi, E. Malaria: An overview. Infect. Drug Resistance. 16, 3339\u201347 (2023).","journal-title":"Infect. Drug Resistance."},{"issue":"1","key":"16972_CR3","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s40101-020-00251-9","volume":"40","author":"S Sato","year":"2021","unstructured":"Sato, S. Plasmodium\u2014a brief introduction to the parasites causing human malaria and their basic biology. J. Physiol. Anthropol. 40(1), 1 (2021).","journal-title":"J. Physiol. Anthropol."},{"issue":"1","key":"16972_CR4","doi-asserted-by":"publisher","first-page":"a025569","DOI":"10.1101\/cshperspect.a025569","volume":"8","author":"DA Milner","year":"2018","unstructured":"Milner, D. A. Malaria pathogenesis. Cold Spring Harb. Perspect. Med. 8(1), a025569 (2018).","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"16972_CR5","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12936-018-2318-1","volume":"17","author":"MT White","year":"2018","unstructured":"White, M. T. et al. Plasmodium vivax and Plasmodium falciparum infection dynamics: Re-infections, recrudescences and relapses. Malar. J. 17, 1\u201315 (2018).","journal-title":"Malar. J."},{"issue":"2","key":"16972_CR6","doi-asserted-by":"publisher","first-page":"695","DOI":"10.3390\/ijms25020695","volume":"25","author":"A Calderaro","year":"2024","unstructured":"Calderaro, A., Piccolo, G. & Chezzi, C. The laboratory diagnosis of malaria: A focus on the diagnostic assays in non-endemic areas. Int. J. Mol. Sci. 25(2), 695 (2024).","journal-title":"Int. J. Mol. Sci."},{"issue":"3","key":"16972_CR7","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.1002380","volume":"14","author":"J Hemingway","year":"2016","unstructured":"Hemingway, J. et al. Tools and strategies for malaria control and elimination: What do we need to achieve a grand convergence in malaria?. PLoS Biol. 14(3), e1002380 (2016).","journal-title":"PLoS Biol."},{"issue":"8","key":"16972_CR8","doi-asserted-by":"publisher","first-page":"1534","DOI":"10.1021\/acsinfecdis.3c00087","volume":"9","author":"H Wei","year":"2023","unstructured":"Wei, H. et al. Rapid and ultrasensitive detection of Plasmodium spp. parasites via the RPA-CRISPR\/Cas12a platform. ACS Infect. Dis. 9(8), 1534\u201345 (2023).","journal-title":"ACS Infect. Dis."},{"key":"16972_CR9","doi-asserted-by":"publisher","first-page":"107350","DOI":"10.1016\/j.actatropica.2024.107350","volume":"258","author":"LH Pati\u00f1o","year":"2024","unstructured":"Pati\u00f1o, L. H. et al. Validation of real-time PCR assays for detecting Plasmodium and Babesia DNA species in blood samples. Acta Trop. 258, 107350 (2024).","journal-title":"Acta Trop."},{"issue":"2","key":"16972_CR10","first-page":"89","volume":"53","author":"MF Rojas","year":"2021","unstructured":"Rojas, M. F. et al. Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus. Rev. Argent Microbiol. 53(2), 89\u201397 (2021).","journal-title":"Rev. Argent Microbiol."},{"issue":"3","key":"16972_CR11","doi-asserted-by":"publisher","first-page":"e0120210","DOI":"10.1371\/journal.pone.0120210","volume":"10","author":"W Xu","year":"2015","unstructured":"Xu, W. et al. SYBR Green real-time PCR-RFLP assay targeting the Plasmodium cytochrome B gene\u2013a highly sensitive molecular tool for malaria parasite detection and species determination. PLoS ONE 10(3), e0120210 (2015).","journal-title":"PLoS ONE"},{"key":"16972_CR12","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12936-021-03662-w","volume":"20","author":"A Lamien-Meda","year":"2021","unstructured":"Lamien-Meda, A., Fuehrer, H.-P., Leitsch, D. & Noedl, H. A powerful qPCR-high resolution melting assay with taqman probe in Plasmodium species differentiation. Malar. J. 20, 1\u20138 (2021).","journal-title":"Malar. J."},{"issue":"1","key":"16972_CR13","doi-asserted-by":"publisher","first-page":"1674","DOI":"10.1038\/s41598-018-36515-9","volume":"9","author":"E Murillo","year":"2019","unstructured":"Murillo, E., Muskus, C., Agudelo, L. A., V\u00e9lez, I. D. & Ruiz-Lopez, F. A new high-resolution melting analysis for the detection and identification of Plasmodium in human and Anopheles vectors of malaria. Sci. Rep. 9(1), 1674 (2019).","journal-title":"Sci. Rep."},{"key":"16972_CR14","first-page":"52839","volume":"105","author":"R Daniels","year":"2015","unstructured":"Daniels, R. et al. Methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria. J. Vis. Exp. 105, 52839 (2015).","journal-title":"J. Vis. Exp."},{"issue":"1","key":"16972_CR15","doi-asserted-by":"publisher","first-page":"15671","DOI":"10.1038\/srep15671","volume":"5","author":"KH Chua","year":"2015","unstructured":"Chua, K. H. et al. Development of high resolution melting analysis for the diagnosis of human malaria. Sci. Rep. 5(1), 15671 (2015).","journal-title":"Sci. Rep."},{"issue":"11","key":"16972_CR16","doi-asserted-by":"publisher","first-page":"e0260087","DOI":"10.1371\/journal.pone.0260087","volume":"16","author":"BIDS Ferreira","year":"2021","unstructured":"Ferreira, B. I. D. S. et al. Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting. PLoS One. 16(11), e0260087 (2021).","journal-title":"PLoS One."},{"key":"16972_CR17","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/1475-2875-12-264","volume":"12","author":"XT Goh","year":"2013","unstructured":"Goh, X. T. et al. Increased detection of Plasmodium knowlesi in Sandakan division, Sabah as revealed by PlasmoNex\u2122. Malar J. 12, 1\u201314 (2013).","journal-title":"Malar J."},{"key":"16972_CR18","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/1475-2875-13-48","volume":"13","author":"SK Mekonnen","year":"2014","unstructured":"Mekonnen, S. K., Aseffa, A., Medhin, G., Berhe, N. & Velavan, T. P. Re-evaluation of microscopy confirmed Plasmodium falciparum and Plasmodium vivax malaria by nested PCR detection in southern Ethiopia. Malar J. 13, 1\u20138 (2014).","journal-title":"Malar J."},{"issue":"2","key":"16972_CR19","doi-asserted-by":"publisher","first-page":"183","DOI":"10.4269\/ajtmh.1999.60.183","volume":"60","author":"JM Rubio","year":"1999","unstructured":"Rubio, J. M. et al. Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea. Am. J. Trop. Med. Hyg. 60(2), 183\u2013187 (1999).","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"16972_CR20","first-page":"365","volume-title":"Methods in Molecular Biology","author":"S Rozen","year":"2000","unstructured":"Rozen, S. & Skaletsky, H. Primer3: Bioinformatics methods and protocols. In Methods in Molecular Biology (eds Krawetz, S. & Misener, S.) 365\u201386 (Totowa, Humana Press, 2000)."},{"issue":"1","key":"16972_CR21","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1016\/j.ympev.2008.01.031","volume":"47","author":"Y Nishimoto","year":"2008","unstructured":"Nishimoto, Y. et al. Evolution and phylogeny of the heterogeneous cytosolic SSU rRNA genes in the genus Plasmodium. Mol. Phylogenet. Evol. 47(1), 45\u201353 (2008).","journal-title":"Mol. Phylogenet. Evol."},{"key":"16972_CR22","doi-asserted-by":"publisher","first-page":"1362","DOI":"10.3389\/fgene.2019.01362","volume":"10","author":"L Gonz\u00e1lez-Cer\u00f3n","year":"2020","unstructured":"Gonz\u00e1lez-Cer\u00f3n, L. et al. Ookinete-specific genes and 18S SSU rRNA evidenced in plasmodium vivax selection and adaptation by sympatric vectors. Front. Genet. 10, 1362 (2020).","journal-title":"Front. Genet."},{"issue":"3","key":"16972_CR23","first-page":"512","volume":"10","author":"K Tamura","year":"1993","unstructured":"Tamura, K. & Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10(3), 512\u2013526 (1993).","journal-title":"Mol. Biol. Evol."},{"issue":"7","key":"16972_CR24","doi-asserted-by":"publisher","first-page":"1870","DOI":"10.1093\/molbev\/msw054","volume":"33","author":"S Kumar","year":"2016","unstructured":"Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33(7), 1870\u20134 (2016).","journal-title":"Mol. Biol. Evol."},{"key":"16972_CR25","doi-asserted-by":"publisher","first-page":"429","DOI":"10.1186\/1475-2875-13-429","volume":"13","author":"PN Kipanga","year":"2014","unstructured":"Kipanga, P. N. et al. High-resolution melting analysis reveals low Plasmodium parasitaemia infections among microscopically negative febrile patients in western Kenya. Malar J. 13, 429 (2014).","journal-title":"Malar J."},{"issue":"2","key":"16972_CR26","doi-asserted-by":"publisher","first-page":"99","DOI":"10.3347\/kjp.2010.48.2.99","volume":"48","author":"A Genc","year":"2010","unstructured":"Genc, A., Eroglu, F. & Koltas, I. S. Detection of Plasmodium vivax by nested PCR and real-time PCR. Korean J Parasitol. 48(2), 99\u2013103 (2010).","journal-title":"Korean J Parasitol."},{"key":"16972_CR27","doi-asserted-by":"publisher","first-page":"687","DOI":"10.4269\/ajtmh.1999.60.687","volume":"60","author":"B Singh","year":"1999","unstructured":"Singh, B. et al. A genus-and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am. J. Trop. Med. Hyg. 60, 687\u2013692 (1999).","journal-title":"Am. J. Trop. Med. Hyg."},{"issue":"4_Suppl","key":"16972_CR28","doi-asserted-by":"publisher","first-page":"68","DOI":"10.4269\/ajtmh.21-1288","volume":"107","author":"A Wesolowski","year":"2022","unstructured":"Wesolowski, A. et al. Policy implications of the southern and central africa international center of excellence for malaria research: Ten years of malaria control impact assessments in hypo-, meso-, and holoendemic transmission zones in Zambia and Zimbabwe. Am. J. Trop. Med. Hyg. 107(4_Suppl), 68\u201374 (2022).","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"16972_CR29","doi-asserted-by":"publisher","first-page":"e110202","DOI":"10.5812\/archcid.110202","volume":"16","author":"Z Yassin","year":"2021","unstructured":"Yassin, Z., Shirvani, F. & Fattahi, M. Comparison of direct sequencing with real-time PCR high resolution melt and PCR restriction fragment length polymorphism analysis to identify clinically important candida species. Archiv. Clin. Infect. Dis. 16, e110202 (2021).","journal-title":"Archiv. Clin. Infect. Dis."},{"key":"16972_CR30","doi-asserted-by":"crossref","unstructured":"Stubbs, S., Blacklaws, B., Yohan, B., Yudhaputri, F. A., Schwem, B., Salva\u00f1a, E. M., et al. A Nanopore-based method for generating complete coding region sequences of dengue virus in resource-limited settings. bioRxiv. 499111 (2018).","DOI":"10.1101\/499111"},{"key":"16972_CR31","first-page":"11","volume":"1","author":"J Tan","year":"2022","unstructured":"Tan, J. & Lau, Y. Diagnostic methods for Plasmodium knowlesi: Performance, limitations and recommendations. Diagn. Ther. 1, 11\u201322 (2022).","journal-title":"Diagn. Ther."},{"issue":"3","key":"16972_CR32","first-page":"459","volume":"30","author":"C Anthony","year":"2013","unstructured":"Anthony, C., Mahmud, R., Lau, Y. L., Syedomar, S. F. & Sri La Sri Ponnampalavanar, S. Comparison of two nested PCR methods for the detection of human malaria. Trop. Biomed. 30(3), 459\u201366 (2013).","journal-title":"Trop. Biomed."},{"issue":"19","key":"16972_CR33","doi-asserted-by":"publisher","first-page":"11150","DOI":"10.3390\/ijms231911150","volume":"23","author":"H Frickmann","year":"2022","unstructured":"Frickmann, H. et al. Metagenomic sequencing for the diagnosis of Plasmodium spp. with different levels of parasitemia in EDTA blood of malaria patients-a proof-of-principle assessment. Int. J. Mol. Sci. 23(19), 11150 (2022).","journal-title":"Int. J. Mol. Sci."},{"issue":"2","key":"16972_CR34","doi-asserted-by":"publisher","first-page":"336","DOI":"10.4269\/ajtmh.15-0569","volume":"94","author":"Y-L Lau","year":"2016","unstructured":"Lau, Y.-L., Lai, M.-Y., Fong, M.-Y., Jelip, J. & Mahmud, R. Loop-mediated isothermal amplification assay for identification of five human Plasmodium species in Malaysia. Am. J. Trop. Med. Hyg. 94(2), 336 (2016).","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"16972_CR35","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1186\/s12936-015-0542-5","volume":"14","author":"PC Lee","year":"2015","unstructured":"Lee, P. C. et al. Molecular detection of human Plasmodium species in Sabah using PlasmoNex\u2122 multiplex PCR and hydrolysis probes real-time PCR. Malar J. 14, 28 (2015).","journal-title":"Malar J."},{"issue":"1","key":"16972_CR36","doi-asserted-by":"publisher","first-page":"28","DOI":"10.4269\/ajtmh.14-0309","volume":"92","author":"YL Lau","year":"2015","unstructured":"Lau, Y. L. et al. Comparison of three molecular methods for the detection and speciation of five human Plasmodium species. Am J Trop Med Hyg. 92(1), 28\u201333 (2015).","journal-title":"Am J Trop Med Hyg."},{"issue":"11","key":"16972_CR37","doi-asserted-by":"publisher","first-page":"1434","DOI":"10.1093\/infdis\/jix507","volume":"216","author":"C Koepfli","year":"2017","unstructured":"Koepfli, C. et al. Sustained malaria control over an 8-year period in Papua New Guinea: The challenge of low-density asymptomatic plasmodium infections. J. Infect. Dis. 216(11), 1434\u20131443 (2017).","journal-title":"J. Infect. Dis."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-16972-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-16972-9","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-16972-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T22:03:26Z","timestamp":1759788206000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-16972-9"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,2]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["16972"],"URL":"https:\/\/doi.org\/10.1038\/s41598-025-16972-9","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,2]]},"assertion":[{"value":"28 April 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"20 August 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"2 October 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"34360"}}